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Background: SARS‐CoV‐2 has triggered a pandemic and contributes to long‐lasting morbidity. Several studies have investigated immediate cellular and humoral immune responses during acute infection. However, little is known about long‐term effects of COVID‐19 on the immune system. Methods: We performed a longitudinal investigation of cellular and humoral immune parameters in 106 non‐vaccinated subjects ten weeks (10 w) and ten months (10 m) after their first SARS‐CoV‐2 infection. Peripheral blood immune cells were analyzed by multiparametric flow cytometry, serum cytokines were examined by multiplex technology. Antibodies specific for the Spike protein (S), the receptor‐binding domain (RBD) and the nucleocapsid protein (NC) were determined. All parameters measured 10 w and 10 m after infection were compared with those of a matched, noninfected control group (n = 98). Results: Whole blood flow cytometric analyses revealed that 10 m after COVID‐19, convalescent patients compared to controls had reduced absolute granulocyte, monocyte, and lymphocyte counts, involving T, B, and NK cells, in particular CD3+CD45RA+CD62L+CD31+ recent thymic emigrant T cells and non‐class‐switched CD19+IgD+CD27+ memory B cells. Cellular changes were associated with a reversal from Th1‐ to Th2‐dominated serum cytokine patterns. Strong declines of NC‐ and S‐specific antibody levels were associated with younger age (by 10.3 years, p <.01) and fewer CD3−CD56+ NK and CD19+CD27+ B memory cells. Changes of T‐cell subsets at 10 m such as normalization of effector and Treg numbers, decline of RTE, and increase of central memory T cell numbers were independent of antibody decline pattern. Conclusions: COVID‐19 causes long‐term reduction of innate and adaptive immune cells which is associated with a Th2 serum cytokine profile. This may provide an immunological mechanism for long‐term sequelae after COVID‐19. [ABSTRACT FROM AUTHOR]

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future. [ABSTRACT FROM AUTHOR]

Background: SARS‐CoV‐2 has triggered a pandemic that is now claiming many lives. Several studies have investigated cellular immune responses in COVID‐19‐infected patients during disease but little is known regarding a possible protracted impact of COVID‐19 on the adaptive and innate immune system in COVID‐19 convalescent patients. Methods: We used multiparametric flow cytometry to analyze whole peripheral blood samples and determined SARS‐CoV‐2‐specific antibody levels against the S‐protein, its RBD‐subunit, and viral nucleocapsid in a cohort of COVID‐19 convalescent patients who had mild disease ~10 weeks after infection (n = 109) and healthy control subjects (n = 98). Furthermore, we correlated immunological changes with clinical and demographic parameters. Results: Even ten weeks after disease COVID‐19 convalescent patients had fewer neutrophils, while their cytotoxic CD8+ T cells were activated, reflected as higher HLA‐DR and CD38 expression. Multiparametric regression analyses showed that in COVID‐19‐infected patients both CD3+CD4+ and CD3+CD8+ effector memory cells were higher, while CD25+Foxp3+ T regulatory cells were lower. In addition, both transitional B cell and plasmablast levels were significantly elevated in COVID‐19‐infected patients. Fever (duration, level) correlated with numbers of central memory CD4+ T cells and anti‐S and anti‐RBD, but not anti‐NC antibody levels. Moreover, a "young immunological age" as determined by numbers of CD3+CD45RA+CD62L+CD31+ recent thymic emigrants was associated with a loss of sense of taste and/or smell. Conclusion: Acute SARS‐CoV‐2 infection leaves protracted beneficial (ie, activation of T cells) and potentially harmful (ie, reduction of neutrophils) imprints in the cellular immune system in addition to induction of specific antibody responses. [ABSTRACT FROM AUTHOR]

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes. [ABSTRACT FROM AUTHOR]

Keywords: COVID-19; immune complex; molecular interaction assay; protective antibodies; SARS-CoV-2; vaccine EN COVID-19 immune complex molecular interaction assay protective antibodies SARS-CoV-2 vaccine 878 883 6 03/09/21 20210301 NES 210301 Abbreviations ACE2 angiotensin-converting enzyme 2 COVID-19 coronavirus disease ELISA enzyme-linked immunosorbent assay HRP horseradish peroxidase IgG, IgA, IgM immunoglobulin G, A, M MERS middle east respiratory syndrome OD optical density RBD receptor-binding domain RNA ribonucleic acid RT-PCR reverse transcription polymerase chain reaction RV rhinovirus S spike protein S1 spike protein receptor-binding subunit S2 spike protein membrane fusion subunit SARS severe acute respiratory syndrome After the appearance of first cases in Wuhan, China in December 2019, the novel human coronavirus disease, COVID-19, has become the first coronavirus pandemic in history.1 On 16 July 2020, more than 13.5 million patients worldwide have been infected with the novel coronavirus, SARS-CoV-2, and more than 584.000 global deaths related to COVID-19 have been reported (see: The Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, Baltimore: https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6). However, in 2002, severe acute respiratory syndrome (SARS) (Group IIb) and, in 2012, Middle East respiratory syndrome (MERS) (Group IIc) were shown to be caused by the novel coronaviruses, SARS-CoV and MERS-CoV, respectively, which caused high death rates in up to 10% of infected people.1 Like SARS-CoV, SARS-CoV-2 uses angiotensin-converting enzyme 2, ACE2 on human cells as its receptor2 and binds to it with its receptor-binding domain (RBD). It has been shown that COVID-19 patients develop SARS-CoV-2-specific antibodies but it is not known if and in how many infected subjects the virus-induced antibodies are protective. [Extracted from the article]

Nuclear factor 'κ‐light‐chain‐enhancer' of activated B cells (NF‐κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR‐derived signals, thereby maintaining their suppressive features. However, the role of NF‐κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF‐κB proteins p50, p65, and c‐Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF‐κB signaling in FOXP3‐transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF‐κB signaling. Additionally, repression of residual NF‐κB signaling in Treg further enhances interleukin‐10 (IL‐10) production. Blockade of NF‐κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF‐κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg‐specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF‐κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy. [ABSTRACT FROM AUTHOR]

Background: In high‐risk populations, allergen‐specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus‐like nanoparticles (VNP) expressing surface‐exposed or shielded allergens. Methods: Full‐length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results: Virus‐like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface‐exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1‐specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen‐specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface‐exposed but not shielded allergen induced allergen‐specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen‐protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg‐dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion: Virus‐like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization. Allergen‐expressing VNP, which are produced in HEK 293T cells with the help of MoMLV structural proteins, are unable to stimulate allergen‐specific B cells or sensitized effector cells (basophils, mast cells). Instead, upon i.n. application, they are being taken up by lung‐resident CD103+ DC and alveolar macrophages, which unpack their (allergic) cargo, expand Foxp3+ Treg cells and prevent from sensitization. [ABSTRACT FROM AUTHOR]

A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here determined whether systemically toxic cytokines such as IL-2 can be restricted to the site of antigen presentation and used as ‘natural adjuvants’. Therefore, we devised antigen-presenting virus-like nanoparticles (VNP) co-expressing IL-2 attached to different membrane-anchors and assessed their potency to modulate CD8+ T cell responses in vitro and in vivo. Efficient targeting of IL-2 to lipid rafts and ultimately VNP was achieved by fusing IL-2 at its C-terminus to a minimal glycosylphosphatidylinositol (GPI)-anchor acceptor sequence. To identify optimal membrane-anchor dimensions we inserted one (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b between IL-2 and the minimal GPI-anchor acceptor sequence of CD16b (GPI). We found that the 2IgGPI version was superior to all other evaluated IL-2 variants (IL-2v) in terms of its i) degree of targeting to lipid rafts and to the VNP surface, ii) biological activity, iii) co-stimulation of cognate T cells in the absence of bystander activation and iv) potency to induce differentiation and acquisition of CD8+ T cell effector functions in vitro and in vivo. In contrast, the GPI version rather favored memory precursor cell formation. These results exemplify novel beneficial features of membrane-bound IL-2, which in addition to its mere T cell stimulatory capacity include the induction of differential effector and memory functions in CD8+ T lymphocytes. [ABSTRACT FROM AUTHOR]

Chloroquine (CQ) is widely used as an anti-inflammatory therapeutic for rheumatic diseases. Although its modes of action on the innate immune system are well described, there is still insufficient knowledge about its direct effects on the adaptive immune system. Thus, we evaluated the influence of CQ on activation parameters of human CD4+ T-cells. CQ directly suppressed proliferation, metabolic activity and cytokine secretion of T-cells following anti-CD3/anti-CD28 activation. In contrast, CQ showed no effect on up-regulation of T-cell activation markers. CQ inhibited activation of all T helper cell subsets, although IL-4 and IL-13 secretion by Th2 cells were less influenced compared to other Th-specific cytokines. Up to 10 μM, CQ did not reduce cell viability, suggesting specific suppressive effects on T-cells. These properties of CQ were fully reversible in re-stimulation experiments. Analyses of intracellular signaling showed that CQ specifically inhibited autophagic flux and additionally activation of AP-1 by reducing phosphorylation of c-JUN. This effect was mediated by inhibition of JNK catalytic activity. In summary, we characterized selective and reversible immunomodulatory effects of CQ on human CD4+ T-cells. These findings provide new insights into the biological actions of JNK/AP-1 signaling in T-cells and may help to expand the therapeutic spectrum of CQ. [ABSTRACT FROM AUTHOR]

Background: Treg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development., Objective: To evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds., Materials and Methods: We used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds., Results: We show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils., Conclusions: BX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases., Competing Interests: With regards to the authors disclosure of potential conflicts of interest we would like to indicate that WP received honoraria from Novartis, Astra Zeneca and Roche, GSK and Bristol-Myers Squibb outside the submitted work and GG reports receiving personal fees from Bencard, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tauber, Kratzer, Schatzlmaier, Smole, Köhler, Rausch, Kranich, Trapin, Neunkirchner, Zabel, Jutz, Steinberger, Gadermaier, Brocker, Stockinger, Derdak and Pickl.)

Background: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important., Methods: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus., Results: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG 1 , and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants., Conclusion: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2-neutralizing antibodies conferring sterilizing immunity., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Chloroquine (CQ) is widely used as an anti-inflammatory therapeutic for rheumatic diseases. Although its modes of action on the innate immune system are well described, there is still insufficient knowledge about its direct effects on the adaptive immune system. Thus, we evaluated the influence of CQ on activation parameters of human CD4 + T-cells. CQ directly suppressed proliferation, metabolic activity and cytokine secretion of T-cells following anti-CD3/anti-CD28 activation. In contrast, CQ showed no effect on up-regulation of T-cell activation markers. CQ inhibited activation of all T helper cell subsets, although IL-4 and IL-13 secretion by Th2 cells were less influenced compared to other Th-specific cytokines. Up to 10 μM, CQ did not reduce cell viability, suggesting specific suppressive effects on T-cells. These properties of CQ were fully reversible in re-stimulation experiments. Analyses of intracellular signaling showed that CQ specifically inhibited autophagic flux and additionally activation of AP-1 by reducing phosphorylation of c-JUN. This effect was mediated by inhibition of JNK catalytic activity. In summary, we characterized selective and reversible immunomodulatory effects of CQ on human CD4 + T-cells. These findings provide new insights into the biological actions of JNK/AP-1 signaling in T-cells and may help to expand the therapeutic spectrum of CQ., Competing Interests: The authors declare no competing financial interests.