The COVID-19 mRNA vaccine Comirnaty induces anaphylactic shock in an anti-PEG hyperimmune large animal model: Role of complement in cardiovascular, hematological, and inflammatory mediator changes (preprint)

Background: Comirnaty, Pfizer-BioNTech's polyethylene-glycol (PEG)-containing Covid-19 vaccine, can cause hypersensitivity reactions (HSRs) in a small fraction of immunized people which can, very rarely, culminate in life-threatening anaphylaxis. A role of anti-PEG antibodies (Abs) has been proposed, but causality has not yet been proven in an animal model. This study aimed to provide such evidence using anti-PEG hyperimmune pigs (i.e., pigs displaying very high levels of anti-PEG Abs). We also sought to find evidence for the role of complement (C) activation and thromboxane A2 (TXA2) release in blood as contributing effects to anaphylaxis. Methods: Pigs (n=6) were immunized with 0.1 mg/kg PEGylated liposome (Doxebo) i.v. the rise of anti-PEG IgG and IgM was measured in serial blood samples with ELISA. After 2-3 weeks, during the height of seroconversion, the animals were injected i.v. with 1/3 human vaccine dose (HVD) of Comirnaty, and the hemodynamic (PAP, SAP), cardiopulmonary (HR, EtCO2,), hematological parameters (WBC, granulocyte, lymphocyte, and platelet counts) and blood immune mediators (anti-PEG IgM and IgG Abs, C3a and TXA2) were measured as endpoints of HSRs. Results: A week after immunization of 6 pigs with Doxebo, the level of anti-PEG IgM and IgG rose 5-10-thousands-fold in all animals, and they all developed anaphylactic shock to i.v. injection of 1/3 HVD of Comirnaty. The reaction, starting within 1 min, led to the abrupt decline of SAP along with maximal pulmonary hypertension, decreased pulse pressure amplitude, tachycardia, granulo- and thrombocytopenia, and paralleling rises of plasma C3a and TXB2 levels. These vaccine effects were not observed in non-immunized pigs. Conclusions: Consistent with previous studies with PEGylated nano-liposomes, these data show a causal role of anti-PEG Abs in the anaphylaxis to Comirnaty. The reaction involves C activation, and, hence, it represents C activation-related pseudo-allergy (CARPA). The setup provides the first large-animal model for mRNA-vaccine-induced anaphylaxis in humans.

Comparison of morphological and metabolic imaging of COVID-19 pneumonia in a prospective clinical study (preprint)

Purpose To evaluate morphological and metabolic findings in novel coronavirus 19 disease (COVID-19) with 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (FDG-PET/CT). Materials and methods This was a single-centre, prospective clinical trial enrolling consecutive patients who required hospitalisation due to COVID-19 infection. All patients underwent routine chest CT on admission and a follow-up FDG-PET/CT scan on the 7th day of hospitalisation. COVID-19 related lung alterations, such as ground-glass opacity (GGO) and consolidation were quantified with semi-automated software using deep learning (DL) and metabolic parameters were expressed with PET-based metabolic inflammatory volume (MIV) and total inflammatory activity (TIA). The primary outcome was defined as increased inflammatory state on PET scan, with the median MIV and TIA being the cut-off value. Results Forty-four patients were enrolled (25 men; median [IQR] age: 52 [49-61] years). The median [IQR] MIV and TIA were 209 [73-517] ml and 499 [155-1429], respectively. The percentage of GGO and total lung CT severity scores at baseline CT showed weak correlation with MIV and TIA (r=0.33-0.39; p=0.13-0.34). At follow-up, we detected a strong correlation between all chest CT abnormalities and MIV and TIA (r=0.77; p<0.01 and r=0.75; p<0.01, respectively), as well as between CT severity scores and MIV and TIA (r=0.77; p<0.01 and r=0.75; p<0.01, respectively). Logistic regression analysis adjusted for demographics revealed that the extent of chest CT abnormalities on follow-up was an independent predictor of high inflammatory state (OR [by 1% change] =1.11 for both MIV and TIA; p=0.018 for MIV and p=0.021 for TIA). Also, a model encompassing CT abnormalities, interleukin-6 and lactate-dehydrogenase levels at follow-up showed high predictive values for inflammatory state, with an area-under-the-curve (AUC) on receiver operating characteristics analysis of 0.88. Conclusion The metabolic inflammatory volume and activity of COVID-19-pneumonia showed good correlation with morphological changes on CT imaging performed 7 days after patient hospitalization. Combining CT and laboratory data (lactate dehydrogenase and interleukin-6 levels), FDG-PET-based lung inflammatory status could effectively be predicted. Trial registration: www.clinicaltrials.gov (ID: NCT05009563). Registered 17 August 2021 (retrospectively registered), first patient enrolled: 13 January 2021.

Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions and immunogenicity of PEG-containing Covid-19 vaccines (preprint)

The polyethylene-glycol (PEG)-containing Covid-19 vaccines can cause hypersensitivity reactions (HSRs), or rarely, life-threatening anaphylaxis. A causal role of anti-PEG antibodies (Abs) has been proposed, but not yet proven in humans. The 191 blood donors in this study included 10 women and 5 men who displayed HSRs to Comirnaty or Spikevax Covid-19 vaccines with 3 anaphylaxis. 118 donors had pre-vaccination anti-PEG IgG/IgM values as measured by ELISA, of which >98% were over background regardless of age, indicating the presence of these Abs in almost everyone. Their values varied over 2-3 orders of magnitude and displayed strong left-skewed distribution with 3-4% of subjects having >15-30-fold higher values than the respective median. First, or booster injections with both vaccines led to significant rises of anti-PEG IgG/IgM with >10-fold rises in about ~10% of Comirnaty, and all Spikevax recipients, measured at different times after the injections. The anti-PEG Ab levels measured within 4-months after the HSRs were significantly higher than those in nonreactors. Serial testing of plasma (n=361 tests) showed the SARS-CoV-2 neutralization IgG to vary over a broad range, with a trend for biphasic dose dependence on anti-PEG Abs. The highest prevalence of anti-PEG Ab positivity in human blood reported to date represents new information which can most easily be rationalized by daily exposure to common PEG-containing medications and/or household items. The significantly higher, HSR-non-coincidental blood level of anti-PEG Abs in hypersensitivity reactor vs. non-reactors, taken together with relevant clinical and experimental data in the literature, suggest that anti-PEG Ab supercarrier people might be at increased risk for HSRs to PEG-containing vaccines, which themselves can induce these Abs via bystander immunogenicity. Our data also raise the possibility that anti-PEG Abs might also contribute to the reduction of these vaccines' virus neutralization efficacy. Thus, screening for anti-PEG Ab supercarriers may identify people at risk for HSRs or reduced vaccine effectiveness.

Zymosan-induced leukocyte and cytokine changes in pigs: a new model for streamlined drug testing against severe COVID-19 (preprint)

Injection of 0.1 mg/kg zymosan in pigs i.v. elicited transient hemodynamic disturbance within minutes, without major blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This change was followed by a transient granulopenia with a trough at 1 h, and then, up to about 6 h, a major granulocytosis, resulting in a 3-4-fold increase of neutrophil-to-lymphocyte ratio (NLR). In parallel with the changes in WBC differential, qRT-PCR and ELISA analyses showed increased transcription and/or release of inflammatory cytokines and chemokines into blood, including IL-6, TNF-, CCL-2, CXCL-10, and IL-1RA. The expression of IL-6 peaked at already 1.5-2.5 h, and we observed significant correlation between lymphopenia and IL-6 gene expression. While these changes are consistent with zymosan's known stimulatory effect on both the humoral and cellular arms of the innate immune system, what gives novel clinical relevance to the co-manifestation of above hemodynamic, hematological, and immune changes is that they represent independent bad prognostic indicators in terminal COVID-19 and other diseases involving cytokine storm. Thus, within a 6 h experiment, the model enables consecutive reproduction of a symptom triad that is characteristic of late-stage COVID-19. Given the limitations of modeling cytokine storm in animals and effectively treating severe COVID-19, the presented relatively simple large animal model may advance the R&D of drugs against these conditions. One of these disease markers (NLR), obtained from a routine laboratory endpoint (WBC differential), may also enable streamlining the model for high throughput drug screening.

Soluble Dipeptidyl-Peptidase 4 (sDPP4) Serum Activity Is Associated With the Severity of COVID-19 Disease and is a Strong Prognostic Biomarker of Mortality (preprint)

Background: Type 2 diabetes mellitus (T2DM) and obesity are risk factors of COVID-19 disease course. Retrospective observational analyses reported improvement in COVID-19 severe outcomes and mortality in T2DM patients using a DPP4 inhibitor. However, little is known about the prognostic value of circulating soluble DPP4 activity in patients with SARS-CoV-2 infection.Methods: We conducted an observational, retrospective cohort study with 184 Hungarian adults. Hospitalized patients with acute COVID-19 disease (n=102) and plasma donors (n=43) recovered from prior SARS-CoV-2 infection were enrolled from April to July, 2020 at two institutions in Budapest. “Non-COVID-19 controls” (n=39) were employed with serum sDPP4 activity determined from samples taken before September, 2019. Duplicate serum sDPP4 activity determinations by microplate base enzyme kinetic assay were successful in 182 individuals from different COVID-19 disease severity groups (acute COVID-19 patients were classified into 4 and plasma donors into 2 categories based on peak severity).Findings: A significant difference in sDPP4 activity was found among study groups (p<0·0001): acute SARS-CoV-2 infected group (n=100, median=23·24 U/L [IQR 17·8-30·34]) vs all non-acutely ill participants (n=82, median=35·62 U/L [IQR 31·17-42·09], and plasma donors (median=40·16 U/L [IQR 34·28-49·49], both p-values<0·0001). sDPP4 activity was lower in hospitalized patients with acute SARS-CoV-2 who died (n=24, median=17·08 U/L, [IQR 16·47-22·12]) vs survivors (n=76, median=25·08 U/L [IQR 20·21-30·80], p<0·0001). Logistic regression (p=0·0023) indicated that sDPP4 activity is a prognostic biomarker of mortality. ROC AUC of 73·33% (95%CI: 61·98%-84·68%, p[area=0·5]<0·0001), and 83·45% (95%CI: 74·48%-92·51%, p[area=0·5]<0·0001) characterized the sDPP4 activity test (cut-off 22·25 U/L) and in combination with age in predicting death (median 9·5 days prior) among hospitalized patients with acute COVID-19 disease, respectively.Interpretation: We concluded that serum sDPP4 activity is associated with the severity and a strong biomarker of mortality in COVID-19 disease.Funding Information: Ramgen funded the sDPP4 activity measurement and related costs in the study.Declaration of Interests: Semmelweis University and Central Hospital of Southern Pest, National Institute for Infectious Diseases and Haematology has entered into a contractual partnership with Ramgen to conduct this research and they submitted a patent application (HIPO Ref: 311174000-20210226-1522-004576, inventors: GF, ZP, IVN). ÁN reports a prior fellowship at the Semmelweis University - Rácz Károly Doctoral School which ended prior to the initiation of this project for which he was contracted with Ramgen. IB reports institutional grants or contracts form Envision 2000 and EuroPN (Fresenius Kabi). BL reports payment or honoraria from GlaxoSmithKline, MSD, Fresenius-Kabi, Gilead Sciences, outside of the submitted work. MA reports an associate professor position of Eötvös Loránd University, Faculty of Science, Department of Probability Theory and Statistics, Institute of Mathematics and that he was separately contracted with Ramgen unconnected to his other employments. TM reports on a data safety monitoring board or advisory board participation at AbbVie, BMS, Janssen-Cilag, Novartis, Pfizer, Takeda. CG reports stocks in Ramgen and other interests as co-CEO in Ramgen. BM reports institutional payments from Medtronic, Boston Scientific; personal fees from Biotronik, Abbott, AstraZeneca, Boehringer Ingelheim, and Novartis, outside of the submitted work. PF reports leadership role as President of Hungarian Society for Experimental and Clinical Pharmacology; and other interests as the founder, and CEO of PharmaHungary Group, outside of the submitted work. VNI reports grants or contracts from National Research, Development and Innovation Office, National Research, Development and Innovation Office / Ministry of Innovation and Technology, and Ministry of Human Capacities, outside of the submitted work. GF reports a paid part time employment at the Semmelweis University- Eötvös Loránd Research Network (Molecular Medicine Study group, Hungarian Academy of Sciences, where his employer did not declare employee conflict of interest for this independently conducted project); support for attending meetings and personal fees from 77 Elektronika, and Diabetes and Metabolism Foundation, outside of the submitted work; stocks in Biomerieux and Ramgen and other interests as co-CEO in Ramgen. All the other authors have nothing to declare.Ethics Approval Statement: Study participants signed the informed consent for the whole project and all the sample collections as well as the subsequent study on sDPP4 activity conducted by Ramgen possessed the ethical approval of the national ethical body of Hungary (Medical Research Council Scientific and Research Committee, reference: IV/4403-4/2020/EKU; December 30, 2020). The study was conducted according to the declaration of Helsinki.