mRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions.

A small fraction of people vaccinated with mRNA-lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech's Comirnaty and Moderna's Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1ß < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1ß, and TNF-α, suggesting C-dependence of these cytokines' induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents.

Zymosan Particle-Induced Hemodynamic, Cytokine and Blood Cell Changes in Pigs: An Innate Immune Stimulation Model with Relevance to Cytokine Storm Syndrome and Severe COVID-19.

Hemodynamic disturbance, a rise in neutrophil-to-lymphocyte ratio (NLR) and release of inflammatory cytokines into blood, is a bad prognostic indicator in severe COVID-19 and other diseases involving cytokine storm syndrome (CSS). The purpose of this study was to explore if zymosan, a known stimulator of the innate immune system, could reproduce these changes in pigs. Pigs were instrumented for hemodynamic analysis and, after i.v. administration of zymosan, serial blood samples were taken to measure blood cell changes, cytokine gene transcription in PBMC and blood levels of inflammatory cytokines, using qPCR and ELISA. Zymosan bolus (0.1 mg/kg) elicited transient hemodynamic disturbance within minutes without detectable cytokine or blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This was followed by a transient granulopenia and then, up to 6 h, major granulocytosis, resulting in a 3-4-fold increase in NLR. These changes were paralleled by massive transcription and/or rise in IL-6, TNF-alpha, CCL-2, CXCL-10, and IL-1RA in blood. There was significant correlation between lymphopenia and IL-6 gene expression. We conclude that the presented model may enable mechanistic studies on late-stage COVID-19 and CSS, as well as streamlined drug testing against these conditions.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Complement-mediated hypersensitivity reactions to an amphotericin B-containing lipid complex (Abelcet) in pediatric patients and anesthetized rats: Benefits of slow infusion.

Intravenous administration of lipid-based nanodrugs can cause hypersensitivity, also known as infusion reactions (IRs), that can be attenuated by slow infusion in adult patients. We studied the role of infusion rate and complement (C) activation in IRs in pediatric patients treated with Abelcet, and also in anesthetized rats. IRs were observed in 6 out of 10 (60%) patients who received Abelcet infusion in 4 h or less, while no patients who received the infusion in 6 h showed C activation or IRs. The rat model indicated an inverse relationship between infusion speed and Abelcet-induced hypotension, taken as an experimental endpoint of IRs, while the rise of C3a in blood, an index of C activation, directly correlated with hypotension. The results suggest that pediatric patients are more prone to produce IRs, and that the optimal infusion time of Abelcet may be much longer than the presently recommended 2 h.

A porcine model of hemodialyzer reactions: roles of complement activation and rinsing back of extracorporeal blood.

Hemodialysis reactions (HDRs) resemble complement-activation-related pseudoallergy (CARPA) to certain i.v. drugs, for which pigs provide a sensitive model. On this basis, to better understand the mechanism of human HDRs, we subjected pigs to hemodialysis using polysulfone (FX CorDiax 40, Fresenius) or cellulose triacetate (SureFlux-15UX, Nipro) dialyzers, or Dialysis exchange-set without membranes, as control. Experimental endpoints included typical biomarkers of porcine CARPA; pulmonary arterial pressure (PAP), blood cell counts, plasma sC5b-9 and thromboxane-B2 levels. Hemodialysis (60 min) was followed by reinfusion of extracorporeal blood into the circulation, and finally, an intravenous bolus injection of the complement activator zymosan. The data indicated low-extent steady rise of sC5b-9 along with transient leukopenia, secondary leukocytosis and thrombocytopenia in the two dialyzer groups, consistent with moderate complement activation. Surprisingly, small changes in baseline PAP and plasma thromboxane-B2 levels during hemodialysis switched into 30%-70% sharp rises in all three groups resulting in synchronous spikes within minutes after blood reinfusion. These observations suggest limited complement activation by dialyzer membranes, on which a membrane-independent second immune stimulus was superimposed, and caused pathophysiological changes also characteristic of HDRs. Thus, the porcine CARPA model raises the hypothesis that a second "hit" on anaphylatoxin-sensitized immune cells may be a key contributor to HDRs.

A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression.

BACKGROUND: Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His6- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively. RESULTS: Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His12, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His6, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. CONCLUSIONS: The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases.

One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.

γ-Tubulin interacts with E2F transcription factors to regulate proliferation and endocycling in Arabidopsis.

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it also functions in nuclear processes and in cell division control independently of its canonical role. We found that in Arabidopsis thaliana, γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of their dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F-dependent manner, probably in complex with the E2F-DP heterodimer. The up-regulation of E2F target genes PCNA, ORC2, CDKB1;1, and CCS52A under γ-tubulin silencing suggests a repressive function for γ-tubulin at G1/S and G2/M transitions, and the endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with the E2F-DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development.

Nanostructured lipid carriers accumulate in atherosclerotic plaques of ApoE-/- mice.

Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of 64Cu-fonctionalized NLC in atherosclerotic mice. 64Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure homogenization at tens-of-grams scale. 24 h after injection of 64Cu-chelated particles in ApoE-/- mice, focal regions of the aorta showed accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in aortas isolated from ApoE-/- mice compared to wild type (WT) control (8.95 [7.58, 10.16]×108 vs 4.59 [3.11, 5.03]×108 QL/mm2, P < 0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical translation potential for targeted delivery and imaging purposes in atherosclerosis.

Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

Complement Activation-Related Pathophysiological Changes in Anesthetized Rats: Activator-Dependent Variations of Symptoms and Mediators of Pseudoallergy.

Complement (C) activation can underlie the infusion reactions to liposomes and other nanoparticle-based medicines, a hypersensitivity syndrome that can be partially reproduced in animal models. However, the sensitivities and manifestations substantially differ in different species, and C activation may not be the only cause of pathophysiological changes. In order to map the species variation of C-dependent and -independent pseudoallergy (CARPA/CIPA), here we used known C activators and C activator liposomes to compare their acute hemodynamic, hematological, and biochemical effects in rats. These C activators were cobra venom factor (CVF), zymosan, AmBisome (at 2 doses), its amphotericin B-free vehicle (AmBisombo), and a PEGylated cholesterol-containing liposome (PEG-2000-chol), all having different powers to activate C in rat blood. The pathophysiological endpoints measured were blood pressure, leukocyte and platelet counts, and plasma thromboxane B2, while C activation was assessed by C3 consumption using the Pan-Specific C3 assay. The results showed strong linear correlation between C activation and systemic hypotension, pointing to a causal role of C activation in the hemodynamic changes. The observed thrombocytopenia and leukopenia followed by leukocytosis also correlated with C3 conversion in case of C activators, but not necessarily with C activation by liposomes. These findings are consistent with the double hit hypothesis of hypersensitivity reactions (HSRs), inasmuch as strong C activation can fully account for all symptoms of HSRs, but in case of no-, or weak C activators, the pathophysiological response, if any, is likely to involve other activation pathways.

Characterization of auxin transporter PIN6 plasma membrane targeting reveals a function for PIN6 in plant bolting.

Auxin gradients are sustained by series of influx and efflux carriers whose subcellular localization is sensitive to both exogenous and endogenous factors. Recently the localization of the Arabidopsis thaliana auxin efflux carrier PIN-FORMED (PIN) 6 was reported to be tissue-specific and regulated through unknown mechanisms. Here, we used genetic, molecular and pharmacological approaches to characterize the molecular mechanism(s) controlling the subcellular localization of PIN6. PIN6 localizes to endomembrane domains in tissues with low PIN6 expression levels such as roots, but localizes at the plasma membrane (PM) in tissues with increased PIN6 expression such as the inflorescence stem and nectary glands. We provide evidence that this dual localization is controlled by PIN6 phosphorylation and demonstrate that PIN6 is phosphorylated by mitogen-activated protein kinases (MAPKs) MPK4 and MPK6. The analysis of transgenic plants expressing PIN6 at PM or in endomembrane domains reveals that PIN6 subcellular localization is critical for Arabidopsis inflorescence stem elongation post-flowering (bolting). In line with a role for PIN6 in plant bolting, inflorescence stems elongate faster in pin6 mutant plants than in wild-type plants. We propose that PIN6 subcellular localization is under the control of developmental signals acting on tissue-specific determinants controlling PIN6-expression levels and PIN6 phosphorylation.

Kinase-Associated Phosphoisoform Assay: a novel candidate-based method to detect specific kinase-substrate phosphorylation interactions in vivo.

BACKGROUND: Protein kinases are important components of signalling pathways, and kinomes have remarkably expanded in plants. Yet, our knowledge of kinase substrates in plants is scarce, partly because tools to analyse protein phosphorylation dynamically are limited. Here we describe Kinase-Associated Phosphoisoform Assay, a flexible experimental method for directed experiments to study specific kinase-substrate interactions in vivo. The concept is based on the differential phosphoisoform distribution of candidate substrates transiently expressed with or without co-expression of activated kinases. Phosphorylation status of epitope-tagged proteins is subsequently detected by high-resolution capillary isoelectric focusing coupled with nanofluidic immunoassay, which is capable of detecting subtle changes in isoform distribution. RESULTS: The concept is validated by showing phosphorylation of the known mitogen-activated protein kinase (MAPK) substrate, ACS6, by MPK6. Next, we demonstrate that two transcription factors, WUS and AP2, both of which are shown to be master regulators of plant development by extensive genetic studies, exist in multiple isoforms in plant cells and are phosphorylated by activated MAPKs. CONCLUSION: As plant development flexibly responds to environmental conditions, phosphorylation of developmental regulators by environmentally-activated kinases may participate in linking external cues to developmental regulation. As a counterpart of advances in unbiased screening methods to identify potential protein kinase substrates, such as phosphoproteomics and computational predictions, our results expand the candidate-based experimental toolkit for kinase research and provide an alternative in vivo approach to existing in vitro methodologies.

Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades.

Mitogen-activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organized into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The Arabidopsis mitogen-activated protein kinase 9 (AtMPK9) is a member of an atypical class of MAPKs. Representatives of this MAPK family have a TDY phosphoacceptor site, a long C-terminal extension and lack the common MAPKK-binding docking motif. In the present paper, we describe multiple in vitro and in vivo data showing that AtMPK9 is activated independently of any upstream MAPKKs but rather is activated through autophosphorylation. We mapped the autophosphorylation sites by MS to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase-inactive mutant form of AtMPK9 is not trans-phosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, such as extracellular-signal-regulated kinase (ERK) 7/8, in terms of an MAPKK-independent activation mechanism.

Comparison of complement activation-related pseudoallergy in miniature and domestic pigs: foundation of a validatable immune toxicity model.

Complement activation-related pseudoallergy (CARPA) is an acute adverse immune reaction caused by many nanomedicines. There is a regulatory need for a sensitive and standardizable in vivo predictive assay. While domestic pigs are a sensitive animal model, miniature pigs are favored in toxicological studies yet their utility as a CARPA model has not yet been explored. Herein, we used liposomal doxorubicin and amphotericin B (Doxil/Caelyx and AmBisome), Cremophor EL and zymosan as CARPA triggers to induce reactions in miniature and domestic pigs, and compared the hemodynamic, hematological, biochemical, and skin alterations. The changes observed after administration of the test agents were very similar in both pig strains, suggesting that miniature pigs are a sensitive, reproducible, and, hence, validatable animal model for CARPA regulatory testing. FROM THE CLINICAL EDITOR: With the advances in nanomedicine research, many new agents are now tested for use in clinical setting. Nonetheless, complement activation-related pseudoallergy (CARPA) is a well known phenomenon which can be caused by nanoparticles. In this study, the authors looked at and compared the use of domestic pigs versus miniature pigs as experimental animals for toxicological studies. Their findings confirmed the possible use of miniature pigs for regulatory testing.

Factor H inhibits complement activation induced by liposomal and micellar drugs and the therapeutic antibody rituximab in vitro.

Hypersensitivity reactions to particulate drugs can partly be caused by complement activation and represent a major complication during intravenous application of nanomedicines. Several liposomal and micellar drugs and carriers, and therapeutic antibodies, were shown to activate complement and induce complement activation-related pseudoallergy (CARPA) in model animals. To explore the possible use of the natural complement inhibitor factor H (FH) against CARPA, we examined the effect of FH on complement activation induced by CARPAgenic drugs. Exogenous FH inhibited complement activation induced by the antifungal liposomal Amphotericin-B (AmBisome), the widely used solvent of anticancer drugs Cremophor EL, and the anticancer monoclonal antibody rituximab in vitro. An engineered form of FH (mini-FH) was more potent inhibitor of Ambisome-, Cremophor EL- and rituximab-induced complement activation than FH. The FH-related protein CFHR1 had no inhibitory effect. Our data suggest that FH or its derivatives may be considered in the pharmacological prevention of CARPA. FROM THE CLINICAL EDITOR: Although liposomes and micelles are already in use in the clinical setting as drug carriers, there remains the potential problem of hypersensitivity due to complement activation. In this article, the authors investigated the use of complement inhibitor factor H (FH) on complement activation and showed good efficacy. The results would therefore suggest the potential application of complement inhibitor in the future.

Surprising lack of liposome-induced complement activation by artificial 1,3-diamidophospholipids in vitro.

Cardio-vascular diseases are the main cause of death, emphasizing the need to improve patient treatment and survival. One therapeutic approach is a liposome-based drug carrier system specifically targeting constricted arteries. The recently discovered mechano-sensitive liposomes use hemodynamic shear-stress differences between healthy and constricted blood vessels as trigger for drug release. Liposomes are promising delivery containers but are being recognized as foreign by the immune system. Complement activation as essential factor of the recognition leads to adverse effects. Here, we tested complement activation by liposomes formulated from the artificial phospholipid Pad-PC-Pad in vitro. Surprisingly no complement activation was detected in human sera and porcine plasma. In in vivo experiments with three pigs, neither anaphylactic reactions nor other significant hemodynamic changes were observed even at comparably high liposome doses. The pilot study holds promise for an absence of complement-mediated adverse effects of Pad-PC-Pad liposomes in human. FROM THE CLINICAL EDITOR: A lot of research has been done on new treatment for cardiovascular diseases. Liposome-based carrier systems have also shown promises. In this article, the authors studied the potential risks of complement activation by liposomes in in-vivo experiments. The absence of complement activation by Pad-PC-Pad liposomes may indicate its use in humans.

The Arabidopsis mitogen-activated protein kinase 6 is associated with γ-tubulin on microtubules, phosphorylates EB1c and maintains spindle orientation under nitrosative stress.

Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro. It was the active form of MAP kinase that was enriched with microtubules and followed similar dynamics to γ-tubulin, moving from poles to midzone during the anaphase-to-telophase transition. We found a novel substrate for MPK6, the microtubule plus end protein, EB1c. The mpk6-2 mutant was sensitive to 3-nitro-l-tyrosine (NO2 -Tyr) treatment with respect to mitotic abnormalities, and root cells overexpressing AP2C3 showed defects in chromosome segregation and spindle orientation. Our data suggest that the active form of MAP kinase interacts with γ-tubulin on specific subsets of mitotic microtubules during late mitosis. MPK6 phosphorylates EB1c, but not EB1a, and has a role in maintaining regular planes of cell division under stress conditions.

Aptasensors for viral diagnostics.

Novel viral diagnostic tools need to be affordable, fast, accurate and easy to use with sensitivity and specificity equivalent or superior to current standards. At present, viral diagnostics are based on direct detection of viral components or indirect detection by measuring antibodies generated in response to viral infection. While sensitivity of detection and quantification are still important challenges, we expect major advances from new assay formats and synthetic binding molecules, such as aptamers. Compared to traditional antibody-based detection, aptamers could provide faster adaptation to continuously evolving virus strains and higher discriminating capacity between specific virus serotypes. Aptamers are very stable and easily modifiable, so are ideal molecules for detection and chemical sensing applications. Here, we review the use of aptasensors for detection of viral pathogens and consider the feasibility of aptasensors to become standard devices for point-of-care diagnostics of viruses.

Comirnaty-induced cardiopulmonary distress and other symptoms of complement-mediated pseudo-anaphylaxis in a hyperimmune pig model: Causal role of anti-PEG antibodies.

Background: Comirnaty, Pfizer-BioNTech's polyethylene-glycol (PEG)-containing Covid-19 vaccine, can cause hypersensitivity reactions (HSRs), or rarely, life-threatening anaphylaxis in a small fraction of immunized people. A causal role of anti-PEG antibodies (Abs) has been proposed, but causality has not yet proven in an animal model. The aim of this study was to provide such evidence using pigs immunized against PEG, which displayed very high levels of anti-PEG antibodies (Abs). We also aimed to find evidence for a role of complement activation and thromboxane A2 release in blood to explore the mechanism of anaphylaxis. Methods: Pigs (n = 6) were immunized with 0.1 mg/kg PEGylated liposome (Doxebo) i.v., and the rise of anti-PEG IgG and IgM were measured in serial blood samples with ELISA. After âˆ¼2-3 weeks the animals were injected i.v. with 1/3 human dose of the PEGylated mRNA vaccine, Comirnaty, and the hemodynamic (PAP, SAP) cardiopulmonary (HR, EtCO2,), hematological (WBC, granulocyte, lymphocyte and platelet counts) parameters and blood immune mediators (anti-PEG IgM and IgG antibodies, thromboxane B2, C3a) were measured as endpoints of HSRs (anaphylaxis). Results: The level of anti-PEG IgM and IgG rose 5-10-thousand-fold in all of 6 pigs immunized with Doxebo by day 6, after which time all animals developed anaphylactic shock to i.v. injection of 1/3 human dose of Comirnaty. The reaction, starting within 1 min involved maximal pulmonary hypertension and decreased systemic pulse pressure amplitude, tachycardia, granulo- and thrombocytopenia, and skin reactions (flushing or rash). These physiological changes or their absence were paralleled by C3a and TXB2 rises in blood. Conclusions: Consistent with previous studies, these data show a causal role of anti-PEG Abs in the anaphylaxis to Comirnaty, which involves complement activation, and, hence, it represents C activation-related pseudo-anaphylaxis. The setup provides the first large-animal model for mRNA-vaccine-induced anaphylaxis in humans.