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Significance Vaccines profit from the addition of adjuvants to better and more specifically initiate, amplify, and shape immune responses. Although the number of adjuvant candidates has steadily increased, peaking in the current SARS-CoV-2 pandemic, little is known about their inherent mode of action. Using human blood immune cells, we established a multilayer method to systematically assess the adjuvants’ effects on innate and adaptive immune cells. By employing a multiplex analysis with cells from 30 different donors, we determined important patterns of adjuvant function. Moreover, we demonstrate correlates of an antiviral immune response using a Toll-like receptor 7/8 ligand adjuvant and single-stranded RNA. This knowledge about adjuvants’ distinct immune signatures supports the selection of safe and effective adjuvants for future vaccines., The COVID-19 pandemic highlights the importance of efficient and safe vaccine development. Vaccine adjuvants are essential to boost and tailor the immune response to the corresponding pathogen. To allow for an educated selection, we assessed the effect of different adjuvants on human monocyte-derived dendritic cells (DCs) and their ability to polarize innate and adaptive immune responses. In contrast to commonly used adjuvants, such as aluminum hydroxide, Toll-like receptor (TLR) agonists induced robust phenotypic and functional DC maturation. In a DC-lymphocyte coculture system, we investigated the ensuing immune reactions. While monophosphoryl lipid A synthetic, a TLR4 ligand, induced checkpoint inhibitors indicative for immune exhaustion, the TLR7/8 agonist Resiquimod (R848) induced prominent type-1 interferon and interleukin 6 responses and robust CTL, B-cell, and NK-cell proliferation, which is particularly suited for antiviral immune responses. The recently licensed COVID-19 vaccines, BNT162b and mRNA-1273, are both based on single-stranded RNA. Indeed, we could confirm that the cytokine profile induced by lipid-complexed RNA was almost identical to the pattern induced by R848. Although this awaits further investigation, our results suggest that their efficacy involves the highly efficient antiviral response pattern stimulated by the RNAs’ TLR7/8 activation.

Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: 1) the Rabbit Pyrogen Test (RPT), 2) the Bacterial Endotoxin Test, 3) the Recombinant Factor C test and the Monocyte Activation Test (MAT). Here, we explored the possibility to replace RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cellular source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the set-up of a very sensitive assay (limit of detection, LOD = 0.04 EU/ml; Sensitivity = 0.1 EU/ml). Both quantitative Method A and semi-quantitative Method B were used for data analysis. Our studies revealed that for vaccine without intrinsic pyrogenicity, as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD in order to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current MAT chapter 2.6.30 and propose a re-evaluation of the validity criteria of the Methods A and B, in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.

We aimed to identify statistical predictor variables of lipopolysaccharide (LPS)-induced physical sickness symptoms during the acute and late inflammatory phases using multivariate regression analyses. Data from N = 128 healthy volunteers who received i.v. LPS injection (0.4 or 0.8 ng/kg) or placebo were pooled for analyses. Physical sickness symptoms experienced during the acute (0-6h postinjection) and late (6-24h postinjection) phases were assessed with the validated General-Assessment-of-Side-Effects (GASE) questionnaire. LPS-treated subjects reported significantly more physical sickness symptoms. Physical symptoms during the acute phase were associated with LPS-induced mood impairments and interleukin (IL)-6 increases, explaining 28.5% of variance in GASE scores. During late phase, LPS-induced increases in cortisol and IL-6 plasma concentrations and baseline depression were significant predictor variables, explaining 38.5% of variance. In patients with recurrent or chronic inflammatory states, these factors may act as risk factors ultimately contributing to an exacerbation of sickness symptoms, and should be considered as potential targets for therapeutic strategies.

Clinical data indicate that inflammatory responses differ across sexes, but the mechanisms remain elusive. Herein, we assessed in vivo and ex vivo cytokine responses to bacterial endotoxin in healthy men and women to elucidate the role of systemic and cellular factors underlying sex differences in inflammatory responses. Participants received an i.v. injection of low-dose endotoxin (0.4 ng/kg body mass), and plasma TNF-α and IL-6 responses were analyzed over a period of 6 h. In parallel, ex vivo cytokine production was measured in endotoxin-stimulated blood samples obtained immediately before in vivo endotoxin administration. As glucocorticoids (GCs) play an important role in the negative feedback regulation of the inflammatory response, we additionally analyzed plasma cortisol concentrations and ex vivo GC sensitivity of cytokine production. Results revealed greater in vivo pro-inflammatory responses in women compared with men, with significantly higher increases in plasma TNF-α and IL-6 concentrations. In addition, the endotoxin-induced rise in plasma cortisol was more pronounced in women. In contrast, no sex differences in ex vivo cytokine production and GC sensitivity were observed. Together, these findings demonstrate major differences in in vivo and ex vivo responses to endotoxin and underscore the importance of systemic factors underlying sex differences in the inflammatory response.

Background Inflammation-induced pain amplification and hypersensitivity play a role in the pathophysiology of numerous clinical conditions. Experimental endotoxemia has recently been implemented as model to analyze immune-mediated processes in human pain. In this study, we aimed to analyze dose- and time-dependent effects of lipopolysaccharide (LPS) on clinically-relevant pain models for musculoskeletal and neuropathic pain as well as the interaction among LPS-induced changes in inflammatory markers, pain sensitivity and negative affect. Methods In this randomized, double-blind, placebo-controlled study, healthy male subjects received an intravenous injection of either a moderate dose of LPS (0.8 ng/kg Escherichia coli), low-dose LPS (0.4 ng/kg), or saline (placebo control group). Pressure pain thresholds (PPT), mechanical pain sensitivity (MPS), and cold pain sensitivity (CP) were assessed before and 1, 3, and 6 h post injection to assess time-dependent LPS effects on pain sensitivity. Plasma cytokines (TNF-α, IL-6, IL-8, IL-10) and state anxiety were repeatedly measured before, and 1, 2, 3, 4, and 6 h after injection of LPS or placebo. Results LPS administration induced a systemic immune activation, reflected by significant increases in cytokine levels, body temperature, and negative mood with pronounced effects to the higher LPS dose. Significant decreases of PPTs were observed only 3 h after injection of the moderate dose of LPS (0.8 ng/kg). MPS and CP were not affected by LPS-induced immune activation. Correlation analyses revealed that decreased PPTs were associated with peak IL-6 increases and negative mood. Conclusions Our results revealed widespread increases in musculoskeletal pain sensitivity in response to a moderate dose of LPS (0.8 ng/kg), which correlate both with changes in IL-6 and negative mood. These data extend and refine existing knowledge about immune mechanisms mediating hyperalgesia with implications for the pathophysiology of chronic pain and neuropsychiatric conditions.

Die Sterilitat von parenteral verabreichten Arzneimitteln wird heutzutage als selbstverstandlich vorausgesetzt. In der pharmazeutischen Industrie wurden exakt definierte Verfahren etabliert, die einen effizienten Schutz vor Kontaminationen durch Bakterien und Pilze bieten. Im Gegensatz dazu ist die Gewahrleistung der mikrobiellen Sicherheit der Mehrzahl der Advanced Therapy Medicinal Products (ATMP), insbesondere der Zelltherapeutika, eine bis dato nicht oder hochstens partiell geloste Aufgabe. Sie stellt eine Herausforderung an Hersteller, Behorden und Arzte dar. Eine ganze Reihe der oben genannten Prinzipien ist bei der Herstellung von Zelltherapeutika nicht anwendbar. So ist die Sterilitat von Ausgangsmaterialien nicht garantiert, die bekannten Verfahren zur Sterilisation sind in der Regel nicht anwendbar, sodass auch die Sterilitat der Endprodukte nicht garantiert werden kann. Angesichts extrem kurzer Laufzeiten vieler Zelltherapeutika von bis zu wenigen Stunden liegen die Ergebnisse der etablierten Methoden zur Sterilitatstestung haufig viel zu spat vor. Auserdem kann aus der Sterilitat der Untersuchungsprobe nicht automatisch auf die Sterilitat des gesamten Produktes geschlossen werden. Konventionelle Methoden zur Pyrogentestung sind fur ATMP in den meisten Fallen nicht anwendbar. In dieser Arbeit wird versucht, die wesentlichen Unzulanglichkeiten in puncto Sterilitat und Pyrogenfreiheit neuartiger Therapeutika aufzuzeigen. Moglichkeiten zur Uberwindung dieser Probleme werden diskutiert, und es wird eine Reihe von Losungsvorschlagen unterbreitet.

Impaired mood and increased anxiety represent core symptoms of sickness behavior that are thought to be mediated by pro-inflammatory cytokines. Moreover, excessive inflammation seems to be implicated in the development of mood/affective disorders. Although women are known to mount stronger pro-inflammatory responses during infections and are at higher risk to develop depressive and anxiety disorders compared to men, experimental studies on sex differences in sickness symptoms are scarce. Thus, the present study aimed at comparing physiological and psychological responses to endotoxin administration between men and women. Twenty-eight healthy volunteers (14 men, 14 women) were intravenously injected with a low dose (0.4 ng/kg) of lipopolysaccharide (LPS) and plasma concentrations of cytokines and neuroendocrine factors as well as negative state emotions were measured before and until six hours after LPS administration. Women exhibited a more profound pro-inflammatory response with significantly higher increases in tumor necrosis factor (TNF)-α and interleukin (IL)-6. In contrast, the LPS-induced increase in anti-inflammatory IL-10 was significantly higher in men. The cytokine alterations were accompanied by changes in neuroendocrine factors known to be involved in inflammation regulation. Endotoxin injection induced a significant increase in noradrenaline, without evidence for sex differences. The LPS-induced increase in cortisol was significantly higher in woman, whereas changes in dehydroepiandrosterone were largely comparable. LPS administration also increased secretion of prolactin, but only in women. Despite these profound sex differences in inflammatory and neuroendocrine responses, men and women did not differ in endotoxin-induced alterations in mood and state anxiety or non-specific sickness symptoms. This suggests that compensatory mechanisms exist that counteract the more pronounced inflammatory response in women, preventing an exaggerated sickness response. Disturbance of these compensatory mechanisms by environmental factors such as stress may promote the development of affective disorders in women.

Background Monophosphoryl lipid A (MPLA), a nontoxic TLR4 ligand derived from lipopolysaccharide (LPS), is used clinically as an adjuvant in cancer, hepatitis, and malaria vaccines and in allergen-specific immunotherapy. Nevertheless, its cell-activating effects have not been analyzed in a comprehensive direct comparison including a wide range of different immune cells. Therefore, the objective of this study was the side-by-side comparison of the immune-modulating properties of MPLA and LPS on different immune cells. Methods Immune-activating properties of MPLA and LPS were compared in human monocytes and mast cells (MCs), a mouse endotoxin shock model (ESM), and mouse bone marrow (BM)-derived myeloid dendritic cells (mDCs), T cells (TCs), B cells, and MCs. Results In a mouse in vivo ESM and a human ex vivo monocyte activation test (MAT), MPLA induced the same cytokine secretion pattern as LPS (ESM: IL-6, IL-12, TNF-α; MAT: IL-1β, IL-6, TNF-α), albeit at lower levels. Mouse mDCs and ex vivo isolated B cells stimulated with MPLA required a higher threshold to induce TRIF-dependent cytokine secretion (IL-1β, IL-6, IL-10, and TNF-α) than did LPS-stimulated cells. In mDC:DO11.10 CD4 TC cocultures, stimulation with MPLA, but not with LPS, resulted in enhanced OVA-specific IL-4 and IL-5 secretion from DO11.10 CD4 TCs. Unexpectedly, in both human and mouse MCs, MPLA, unlike LPS, did not elicit secretion of pro-inflammatory cytokines. Conclusions Compared to LPS, MPLA induced a qualitatively similar, but less potent pro-inflammatory immune response, but was unable to activate human or mouse MCs.

The Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 as the causative agent of a severe respiratory disease with a fatality rate of approximately 30%. The high virulence and mortality rate prompted us to analyze aspects of MERS-CoV pathogenesis, especially its interaction with innate immune cells such as antigen-presenting cells (APCs). Particularly, we analyzed secretion of type I and type III interferons (IFNs) by APCs, i.e., B cells, macrophages, monocyte-derived/myeloid dendritic cells (MDDCs/mDCs), and by plasmacytoid dendritic cells (pDCs) of human and murine origin after inoculation with MERS-CoV. Production of large amounts of type I and III IFNs was induced exclusively in human pDCs, which were significantly higher than IFN induction by severe acute respiratory syndrome (SARS)-CoV. Of note, IFNs were secreted in the absence of productive replication. However, receptor binding, endosomal uptake, and probably signaling via Toll-like receptor 7 (TLR7) were critical for sensing of MERS-CoV by pDCs. Furthermore, active transcription of MERS-CoV N RNA and subsequent N protein expression were evident in infected pDCs, indicating abortive infection. Taken together, our results point toward dipeptidyl peptidase 4 (DPP4)-dependent endosomal uptake and subsequent infection of human pDCs by MERS-CoV. However, the replication cycle is stopped after early gene expression. In parallel, human pDCs are potent IFN-producing cells upon MERS-CoV infection. Knowledge of such IFN responses supports our understanding of MERS-CoV pathogenesis and is critical for the choice of treatment options. IMPORTANCE MERS-CoV causes a severe respiratory disease with high fatality rates in human patients. Recently, confirmed human cases have increased dramatically in both number and geographic distribution. Understanding the pathogenesis of this highly pathogenic CoV is crucial for developing successful treatment strategies. This study elucidates the interaction of MERS-CoV with APCs and pDCs, particularly the induction of type I and III IFN secretion. Human pDCs are the immune cell population sensing MERS-CoV but secrete significantly larger amounts of IFNs, especially IFN-α, than in response to SARS-CoV. A model for molecular virus-host interactions is presented outlining IFN induction in pDCs. The massive IFN secretion upon contact suggests a critical role of this mechanism for the high degree of immune activation observed during MERS-CoV infection.

We have recently shown that highly purified lipoteichoic acid (LTA) represents a major immunostimulatory principle of Staphylococcus aureus. In order to test whether this translates to other bacterial species, we extracted and purified LTA from 12 laboratory-grown species. All LTA induced the release of TNF-alpha, IL-1beta, IL-6 and IL-10 in human whole blood. Soluble CD14 (sCD14) inhibited monokine induction by LTA but failed to confer LTA responsiveness for IL-6 and IL-8 release of human umbilical vein endothelial cells (HUVEC). In a competitive LPS-binding protein (LBP) binding assay, the IC(50) of the tested LTA preparations was up to 3,230-fold higher than for LPS. LBP enhanced TNF-alpha release of human peripheral blood mononuclear cells (PBMC) upon LPS but not LTA stimulation. These data demonstrate a differential role for the serum proteins LBP and sCD14 in the recognition of LPS and LTA. Different efficacies of various anti-CD14 antibodies against LPS vs. LTA-induced cytokine release suggest that the recognition sites of CD14 for LPS and LTA are distinct with a partial overlap. While the maximal achievable monokine release in response to LTA was comparable to LPS, all LTA induced significantly less IL-12 and IFN-gamma. IL-12 substitution increased LTA-inducible IFN-gamma release up to 180-fold, suggesting a critical role of poor LTA-inducible IL-12 for IFN-gamma formation. Pretreatment with IFN-gamma rendered galactosamine-sensitized mice sensitive to challenge with LTA. In conclusion, LTA compared to LPS, are weak inducers of IL-12 and subsequent IFN-gamma formation which might explain their lower toxicity in vivo.

Threats of pyrogenicity were discovered more than a century ago. Measures to determine the safety of parenterals and, more recently, medical devices and cell therapies for human use have been in place for 70 years. Currently, there are three testing possibilities available: the Rabbit Pyrogen Test, the Limulus Amebocyte Lysate test (Bacterial Endotoxin Test), and test systems using human whole blood or human monocytes, called Monocyte Activation Test (MAT). The MAT is based on the human fever reaction and thus most closely reflects the human situation. Unfortunately, regulations and testing guidelines are not fully harmonized, despite formal international validation. Furthermore, data showing that the MAT is capable of covering the totality of possible pyrogens relevant to humans were not included in the MAT validations of the last decade. For this review we collate evidence from published literature, unpublished data of our own, and results from the international validation study to show that there is overwhelming scientific evidence to conclude that the whole blood MAT reliably detects non-endotoxin pyrogens. Therefore, further validation exercises do not seem warranted. Erratum: http://www.altex.org/index.php/altex/article/view/377 Addendum: http://www.altex.org/index.php/altex/article/view/322Addendum: doi:10.14573/altex.1410081

Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1beta; IL-6) upon contact with pyrogens. Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached >90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.

Fractionation of commercial preparations of lipoteichoic acids (LTA) by hydrophobic interaction chromatography (HIC) and nuclear magnetic resonance spectroscopy revealed very inhomogeneous compositions and decomposition of the LTA structure: LTA content of the preparations averaged 61% forStreptococcus pyogenes, 16% forBacillus subtilis, and 75% forStaphylococcus aureus. The decomposition was characterized by a loss of glycerophosphate units as well as alanine andN-acetylglucosamine substituents. All preparations contained—to varying degrees—non-LTA, non-lipopolysaccharide (LPS) immunostimulatory components as indicated by their elution profile in HIC, lack of phosphate, and negative Limulus amoebocyte lysate (LAL) test results. After purification, the commercial LTA fromBacillus subtilisandS. pyogenesbut not LTA fromS. aureusinduced the release of tumor necrosis factor alpha, interleukin 1 beta (IL-1β), IL-6, and IL-10 in human blood. While pure LTA are negative in the LAL assay, endotoxin equivalents of more than 10 ng of LPS/mg of LTA were found in the commercial preparations. Taken together, these data indicate that these crude preparations with relatively high endotoxin contamination are not suitable for characterizing the activation of immune cells by LTA.

Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10(-/-), TLR5(-/-) mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5(-/-) mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10(-/-) mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.

The CD14 molecule expressed on monocytes and macrophages is a high-affinity receptor for bacterial lipopolysaccharide (LPS) and hence an important component of the innate immune system. LPS binding protein (LBP) is required to facilitate the binding of LPS to CD14 in vitro and is necessary for the induction of an inflammatory response to LPS in vivo. Here we show that CD14 and LBP can also bind to lipoteichoic acid from the gram-positive bacterium Bacillus subtilis . Although CD14 does not interact with intact B. subtilis organisms, a brief exposure of the bacteria to serum converts them into a form which can bind to CD14 in an LBP-dependent reaction. When serum-pretreated B. subtilis organisms are incubated with the myelomonocytic cell line U937, which expresses CD14, the bacteria are rapidly phagocytosed. The phagocytosis is strictly dependent both on LBP and on CD14. These in vitro results suggest that LBP plays a role in the innate response not only to gram-negative but also to gram-positive infections.

Disadvantages of the regulatory pyrogen test to assure safety of the end-product Human Serum Albumin (HSA) for parenteral use call for the implementation of an alternative test. In the current study, 16 HSA batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test, conventional and endotoxin-specific LAL assay and monocyte activation test (MAT). It was found that all HSA batches were contaminated with (1,3)-beta-glucans, which interfere with the conventional LAL. Endotoxin-specific LAL was not suitable to test HSA due to unacceptable endotoxin recovery. Experiments combining polymyxin B and MAT demonstrated that pyrogenic batches were mainly contaminated with endotoxins. However, endotoxin-specific LAL failed to detect one of them. The contaminating (1,3)-beta-glucans enhanced the MAT/IL-6 response to endotoxin, but not that of MAT/IL-1beta. The endotoxin equivalent concentrations obtained using the IL-6 readout were usually higher than those using IL-1beta, probably owing to the direct induction of IL-6 release from monocytes by (1,3)-beta-glucans. The MAT correlates with the rabbit pyrogen test, providing a higher safety level for pyrogenicity testing of HSA and probably other therapeutic proteins.