mRNA-LNP COVID-19 vaccine lipids induce low level complement activation and production of proinflammatory cytokines: Mechanisms, effects of complement inhibitors, and relevance to adverse reactions (preprint)

Messenger RNA-containing lipid nanoparticles (mRNA-LNPs) enabled widespread COVID-19 vaccination with a small fraction of vaccine recipients displaying acute or sub-acute inflammatory symptoms. The molecular mechanism of these adverse events (AEs) remains undetermined. Here we report that the mRNA-LNP vaccine, Comirnaty, triggers low-level complement (C) activation and production of inflammatory cytokines, which may be key underlying processes of inflammatory AEs. In serum, Comirnaty and the control PEGylated liposome (Doxebo) caused different rises of C split products, C5a, sC5b-9, Bb and C4d, indicating stimulation of the classical pathway of C activation mainly by the liposomes, while a stronger stimulation of the alternative pathway was equal with the vaccine and the liposomes. Spikevax had similar C activation as Comirnaty, but viral or synthetic mRNAs had no such effect. In autologous serum-supplemented peripheral blood mononuclear cell (PBMC) cultures, Comirnaty caused increases in the levels of sC5b-9 and proinflammatory cytokines in the following order: IL-1 < IFN-{gamma} < IL-1{beta} < TNF- < IL-6 < IL-8, whereas heatinactivation of serum prevented the rises of IL-1, IL-1{beta}, and TNF-. Clinical C inhibitors, Soliris and Berinert, suppressed vaccine-induced C activation in serum but did not affect cytokine production when applied individually. These findings suggest that the PEGylated lipid coating of mRNA-LNP nanoparticles can trigger C activation mainly via the alternative pathway, which may be causally related to the induction of some, but not all inflammatory cytokines. While innate immune stimulation is essential for the vaccines efficacy, concurrent production of C- and PBMC-derived inflammatory mediators may contribute to some of the AEs. Pharmacological attenuation of harmful cytokine production using C inhibitors likely requires blocking the C cascade at multiple points.

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.

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.