Episode 296: Immunological reactions to LNP-based mRNA vaccines
In previous episodes, little attention has been given to the side effects of the mRNA vaccines. There is no doubt that the beneficial effects predominate at the population level. The formulation of mRNA in lipid nanoparticles (LNP) is key to their success. However, it is also very clear that these vaccines are more “reactogenic” than traditional viral inactivated or protein vaccines and that not so much the mRNA, but the LN formulation is responsible for most, if not all, side effects. Besides the rare anaphylactic reactions, local inflammation is very common, but also some regional or organ-specific “delayed type hypersensitivity”-like reaction (e.g. COVID arm, myocarditis, neurological). Also systemic reactions (flu-like syndrome) are not uncommon.
There is clearly a need for further optimization of LNP to reduce their pro-inflammatory effect, while maintaining their adjuvant effect. To separate both effects, a clear understanding is needed on how desirable and unwanted effects could be mechanistically distinguished. Unfortunately, we will see that the mechanisms of unwanted effects have only partly been elucidated today.
In Par 1, I briefly summarize two reviews that provide a traditional frame work to analyze the side effects: the most suspected component to induce immune-mediated reactions is poly-ethylene glycol (PEG)
In Par 2, it is shown that vaccination induces IgG and IgM antibodies to PEG, but there is no convincing evidence that they have a negative impact on the response to the Spike antigen or that they are involved in reactogenicity.
In Par 3 Mouri suggests that IgE or positive skin test to PEG might be useful to screen subjects for allergic reactions to the mRNA vaccine. However two large studies in subjects, who had a documented allergic-like reaction to either the vaccine or to PEG or polysorbate failed to show a strong relation. In fact, in the large majority of these subjects the vaccine could be administered without serious problems. This is a puzzling result, as it clearly argues against a classical IgE-based type 1 hypersensitivity reaction.
In Par 4 the alternative hypothesis of so called CARPA (complement-activation related pseudo-allergy) is explained. Pseudo-allergic reactions may totally resemble a classical anaphylaxis, but they are unpredictable and not strictly antigen-dependent: they may occur at the first encounter with the drug and a repeat dose of the same stimulus will not necessarily induce the same reaction. They have been described with various drugs, including liposomal and micelle-solubilized medicines, which are biochemically related to LNP. Experimental evidence that the Pfizer vaccine could induce such pseudo-allergic reaction in a pig model is provided, but I found only one case report with 3 human cases in the literature.
Par 5 takes a step back and looks at the mechanism of innate immune activation by LNP in a nice review. Two mouse studies provide evidence of their capacity to not only induce profound inflammatory reaction, which can even be lethal (at high intranasal doses), but which also decrease vaccine responses and modulate susceptibility to viral and fungal infection, with even transgenerational effects.
Par 1 Two early reviews
Ep 296-1 A: Janos Szebeni Nature Nanotechnology April 2022: Applying lessons learned from nanomedicines.
Schematic of interaction between LNP–mRNA vaccine components and cells at the site of injection along with a hypothetical mechanism of particle distribution to the systemic circulation
Cellular and molecular players responsible for the generation of an immune response to the LNP–mRNA vaccine that leads to both the desirable immunogenicity and, in some individuals, the adverse immune effects.
Only a very small proportion of vaccine recipients developed anaphylaxis. These individuals are currently either excluded from receiving the second dose of mRNA vaccine or the vaccination is done in hospitals under supervision. Studies outlined in this image would help to shed more light on the mechanisms of anaphylaxis and allow the immunization of more individuals.
The available information suggests that excluding anxiety mediated responses, determining whether individuals have conditions known for their higher complement activity (for example, CVID = common variable immune deficiency)) or hypersensitivity (for example, certain types of HLA and mastocytosis) and reviewing allergy history would further help to identify persons at high risk of hypersensitivity reactions, and to develop strategies for safely vaccinating them.
Ep 296-1 B: Ludger Klimek Allergy Feb 2021 Allergenic components of the mRNA-1273 vaccine
The principle of the PEGylated-lipid nanoparticles as a delivery system for the mRNA is illustrated together with the full list of ingredients contained in the vaccine. PEG-2000 and tromethamine/trometamol as potential triggers of allergic reactions are indicated in red and shown on the left side. Substances that are cross-reactive
to PEG, that is, polysorbates, are used in bread, pastry, chewing gum, ice cream, etc
Different ways of exposition to PEG and PEG analogues are illustrated on the right side.
Algorithm for vaccination with the Moderna vaccine mRNA-1273
Par 2 What about IgG and IgM antibodies to PEG?
Ep 296-2 A: Yi Ju ACS Nano 2022 Anti-PEG Antibodies Boosted in Humans by SARS-CoV‑2 Lipid Nanoparticle mRNA Vaccine
- IgG and IgM anti-PEG increase more after Moderna (mRNA-1273) than after Pfizer (BNT162-b2)
Although pre-existing levels of anti-PEG antibody were sex- and age-dependent, the boost level of
anti-PEG antibody following vaccination was not affected by sex or age.
- The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo.
Also binding with the C1q complement component
- PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination.
- PEG antibodies correlated very weakly with increased systemic reactogenicity following two doses of vaccination
Interpretation: Raised anti-PEG may contribute to immune activation and reactogenicity, but the evidence is indirect and weak.
Ep 296-2 B: Guerrini Int J Mol Med Aug 2022 More extensive study, following 3 doses of either Pfizer or Moderna. They confirm the rise of anti-PEG IgM; but not IgG. Rise after Moderna clearly more pronounced
NO IMPACT on the anti-Spike responses and also NOT on reactogenicity.
Ep 296-2 C: Carreno in Vaccine Sept 2022 confirms that anti-PEG is mainly induced after Moderna vaccine and that there is no obvious association between PEG antibodies and adverse reactions
Par 3: IgE to PEG and polysorbate: relation to allergic reactions?
Ep 296-3: Mariko Mouri Allergology Intern June 2022 investigates IgG, IgE and skin test in a small cohort of patients with hypersensitivity.
Subjects with immediate hypersensitivity have increased levels of PEG-specific IgE (A) and IgG (B) and also some increase of IgE and IgG against polysorbate (PS).
IgE against either PEG or PS is clearly associated with a positive skin test.
Conclusion: the results of this study suggest (but do not prove) that PEG is one of the antigens in the allergy to COVID-19 mRNA vaccines, and both PEG-specific IgE and PEG-specific IgG might be useful in diagnosing immediate-type allergy to PEG.
Ep 296-4: Faisal AlMuhizi Allergic reactions to the coronavirus disease 2019 vaccine (ARCOV) study at McGill Uni
44 patients with a history of reaction to COVID mRNA vaccine or to PEG or PS recruited: 40 (90.1%) had reacted to the first vaccine dose, with 18 (45%) of them had anaphylactic reaction.
All patients underwent skin prick test and 5 (11.3%) had a positive test result and were excluded
A total of 39 patients (88.6%) underwent COVID-19 vaccine challenge at the allergy clinic.
Most tolerated the vaccine, with 18 (40.1%) received a single full dose, 20 (45.4%) 2 split doses, and 6 (13.6%) a graded dosing protocol.
Of the 40 patients who reacted to the first dose, 2 had immediate nonsevere allergic reactions to the second dose.
Conclusion: rather reassuring
- Non-immunoglobulin E-mediated or non-immune mechanism was possibly responsible for the first reaction in most patients who tolerated the second dose without issues?
- Low prevalence of PEG positive skin test (only 2 out of 44)
- No evidence for cross-sensitization between PEG and polysorbate.
Ep 296-5: Iris Otani Ann Allergy Asthma Immunol March 2022 COVID-19 vaccine administration in patients with reported reactions to polyethylene glycol- and polysorbate containing therapeutics.
Similar reassuring results on an even larger scale
- 252 with documented or suspected PEG or PS allergy to medication: Only 3 patients from cohort 2 developed mild rash following vaccination.
- 44 patients with acute symptoms following first-dose mRNA COVID-19 vaccine (27 Pfizer, 17 Moderna): 3 developed symptoms (1 requiring epinephrine) ,but they had negative PEG skin test
Conclusion: PEG and polysorbate skin testing did not identify patients at risk for first dose or recurrent reactions to COVID-19 vaccines.
Screening for PEG and polysorbate allergy may only increase vaccine hesitancy without identifying patients at risk for COVID-19 vaccine reaction
Ep 296-6: Cautious, but reassuring advice from CDC
Par 4: Possible alternative mechanism of hypersensitivity = CARPA (complement-activation related pseudo-allergy)
Ep 296-7 A and B: Reviews by Janos Szbeni in Mol Immunol 2014 and by Bo Zhang in Pharmacology 2018
Pseudoallergy and classical IgE mediated hypersensitivity (anaphylaxis) are clinically indistinguishable: angioedema, urticaria, bronchospasm and gastrointestinal signs , together with skin flushing, headache, edema, hypotension and shock.
Pseudo-allergic reactions are unpredictable and not strictly antigen-dependent: they may occur at the first encounter with the drug and a repeat dose of the same stimulus will not necessarily induce the same reaction ( this “waning” is called tachyphylaxis).
Mechanism is often unknown,
- Activation of complement with release of “anafylatoxins” (C3a, C5a), which activate mastocytes and those release histamine, platelet-activating factor (PAF), thromboxanes (TXA), leukotrienes (LT), tryptase etc
- Other non-IgE mechanisms of mast cell activation:
- Endogenous: IgG, , neuropeptides, cytokines, chemokines, and other inflammatory products
- Exogenous: exercise, hyperosmosis…
Table 1 is a list of drugs associated with CARPA and includes opoids, liposomal or micellar drugs, but also non-steroidal anti-inflammatory drugs (NSAID) and traditional Chinese medicines (TCMI).
Ep 296-8: Xin Ron Lim Vaccine Aug 2021 3 cases of presumed pseudo-allergy to Pfizer BNT162b2
- Normal IgM and IgG antibodies, but not IgE antibodies to the Pfizer BNT162b2 vaccine, in all subjects
- Elevated IgG and IgM to PEG as compared to vaccine naïve and “tolerant’ controls.
- High levels of serum anaphylatoxin C3a were observed in all three patients during acute phase
- Tryptase levels, a marker of mast cell activation, were not elevated.
Patient 3 with the highest levels of anti-PEG IgG, IgM, and anti-Pfizer BNT162b2 IgG and IgM exhibited an enhanced Th2 cytokine serum profile (IL-4, IL-10 and IL-33 elevated) (see Table 2)
Ep 296-9: Laslo Deszi Geroscience 2022: A possible animal model for pseudo-allergy to mRNA vaccine: Landrace pigs. They are know to react to IV liposomes with CARPA.
A pseudo-allergic reaction with activation of complement and thromboxane was indeed elicited in 6/14 pigs after intravenous injection of 2-5 fold the human dose of Pfizer BNT162b2.
Not linked to previous Iv or IM administration if the vaccine.
The only correlate: IgM binding to the whole vaccine, used as antigen in an ELISA, was significantly higher
in reactive animals compared to non-reactive ones.
Clearly, the conditions, used in this model, differ from the human vaccination, because the vaccine was administered at a higher dose and intravenously, instead of intramuscularly. The authors argue that the local inflammation induced at the site of injection may be a trigger or that the nanoparticles “leak out” and reach the general circulation, provoking the systemic anaphylactic reaction.
Par 5: Link between adjuvant and inflammatory activity of LNP through Innate immunity
Ep 296-10: Rein Verbeke Immunity Nov 2022 Innate immune mechanisms of mRNA vaccines
Biodistribution and innate immune cell dynamics upon administration of mRNA-iLNP vaccines
(A) Intramuscular administration of nucleosidemodified mRNA-iLNP vaccines results in local inflammation, which recruits neutrophils, monocytes,and various dendritic cell (DC) subsets from the blood to the injection site by production of chemokines and other inflammatory mediators contributing to the extravasation of immune cells.
(B) mRNA-iLNPs and/or antigen-expressing cells are transported to the draining lymph node. The size and surface properties of the particles can impact biodistribution, protein absorption (opsonization), and cellular uptake.
(C) DCs and monocytes/macrophages contribute to antigen presentation and priming of T cells.
(D) T follicular helper (Tfh) cells provide help to B cells in germinal center (GC) reactions in the presence of follicular DCs, leading to affinity maturation. In mice, an important role for iLNP-induced IL-6 was found in the induction of Tfh and GC B cell responses, while type I IFNs promoted CTL responses.
Abbreviations: m1J: N1-methylpseudouridine, iLNP: ionizable lipid nanoparticle, IL: interleukin, IFN:
interferon, ISG: interferon-stimulated gene, CXCL10: C-X-C motif chemokine ligand 10, CD86: cluster of differentiation 86, FDC: follicular dendritic cell, CTL: cytotoxic T lymphocyte, Th1: T helper 1.
Working model of the innate immune mechanisms that contribute to the immunogenicity and reactogenicity of
(A) Uptake of empty iLNPs by innate immune cells and other cell types is sufficient to induce local and systemic inflammation, characterized by the release of pro-inflammatory cytokines such as IL-1b and IL-6.
(B) The incorporation of modified uridines and stringent IVT mRNA purification drastically lowers the recognition of IVT mRNA by TLR3/7/8 and other RNA sensors. These modifications are important to minimize the negative effects of type I IFN-stimulated RNA sensors on protein expression from the antigen-encoding mRNA and to avoid dose-limiting toxicities. The role of the MDA5-IFN-a signaling pathway in inducing CTLs to BNT162b2 in mice suggests residual type I IFN activity of the current generation of mRNA vaccines.
(C) After administration of a second vaccine dose, there is a strong boost in the T cell responses, which is associated with higher IFN-g production. The enhanced activation of myeloid cells and T cells following boosting may reflect a
crosstalk between lymphocytes and myeloid cells.
Abbreviations: IVT, in vitro transcribed; iLNP, ionizable lipid nanoparticle; m1J, N1-methylpseudouridine; NF-kB, nuclear factor-kB; IL-1R, interleukin-1 receptor; IL-1ra, interleukin-1 receptor antagonist; MYD88, myeloid
differentiation primary response protein 88; dsRNA, double-stranded RNA; MDA5, melanoma differentiation-associated protein 5; RIG-I, retinoic acid-inducible gene I; PRR, pattern recognition receptor; IRF, interferon regulatory factor; IFN-I, type I interferon; IFNAR, interferon-a/b receptor; TYK1, leukocyte receptor tyrosine kinase; JAK, janus kinase; STAT, signal transducer and activator of transcription; ISRE, interferon-sensitive response element; PKR, protein kinase R; OAS, 2’-5’-oligoadenylate synthetase; IFNGR, interferon gamma receptor; GAS, gamma interferon activation site; ISG, interferon-stimulated gene; NK, natural killer; CTL, cytotoxic T lymphocyte; Tfh, T follicular helper; GC, germinal center; TLR, toll-like receptor; CXCL10, C-X-C motif chemokine ligand 10; TNF, tumor
Some suggested improvements
- Optimization of protein-coding and non-coding (3’ and 5’ untranslated regions and poly-A tail) sequence of the mRNA, as well as secondary structure formation to maximize antigen expression and adaptive immune responses (without effect on reactogenicity)
- Both the choice of ionizable lipid and the lipid-to-mRNA ratio in the iLNP can be optimized to avoid systemic distribution of mRNA-iLNPs following intramuscular administration, which may be useful as a strategy to minimize systemic adverse events.
- Subcuraneous adminstratiion in stead of intramuscular may increase immunogenicity without increase of inflammation.
Ep 296-11: Sonja Ndeupen iScience Dec 2021 illustrates the pro-inflammatory potential of both intradermal and intranasal administration of the ionzable lipid nanoparticles (iLNP), used in the present mRNA vaccines.
Mice were given 2.5 µg, which is 8 % of the human dose (30 µg), hence very high !!!
- Intradermal injection: massive increase of neutrophils, followed by dendritic cells and macrophages; increase of several inflammatory cytokines including the “pyrogens IL-1 and bIL-6) and chemokines.
- Intranasal administration results in lung inflammation, loss of body weight and even mortality
In the discussion, the authors admit that dose-response experiments should be performed to have a more “realist” picture.
They suggest ( but do not prove) that these innate responses may “somehow amplify” pseudo-allergic reactions (see anaphylactoid CARPA phenomenon in Par 4).
In addition, they speculate that presentation of vaccine antigenic paptides by MHC class I expressing tissue cells (i.e. non-professional antigen-presenting cells) could result in delayed type hypersensitivity reactions, by cytolytic T cells that secrete additional inflammatory cytokines, leading to phenomena such as the “COVID arm”, myocarditis and central nervous system inflammation.
The conclusion is that it will be necessary to strike a balance between positive adjuvant and negative
inflammatory properties as LNP-associated vaccines move forward….
Ep 296-12: Zhen Qin PLoS Pathogens Sept 2022 present another series of mouse studies suggesting that
- Pre-exposure to mRNA-LNPs led to long-term inhibition of the adaptive immune responses, which the use of some other adjuvants could overcome.
- Remarkably innate immune resistance of mice to heterologous infections with influenza virus increased, while resistance to Candida albicans decreased and these acquired immune traits were passed to to their offspring.
Pre-exposure to mRNA-LNPs has long-lasting effect on adaptive immune responses.
A). Experimental model. Animals at the indicated timepoints post-exposure to PBS or eGFP mRNA-LNP were injected into the same spot with PR8 HA (hemagglutining of Influenza virus) mRNA-LNP and then anti-HA responses assessed as depicted two weeks later.
B). Summary AUC graph on serum anti-HA antibody levels.
C). Relative Germinal Center B cell responses from the same mice. Each dot represents a separate mouse.
Innate immune fitness is altered with pre-exposure to mRNA-LNP
A). Experimental model. Two weeks post-exposure to PBS or eGFP mRNA-LNP the animals were infected with sublethal doses of PR8 HA influenza or Candida albicans. The weights and other attributes were monitored as depicted.
Pre-exposure to mRNA-LNP
B) Less body weight drop after PR8 HA influenza infection and C). Much lower viral copies in the lungs.
D) More body weight drop after Candida infection and E). Higher Candida CFU numbers in the kidneys
Immune changes induced by the mRNA-LNP platform can be inherited.
A). Experimental model. Adult WT B6 male and female mice were intradermally immunized with 10 μg of mRNA-LNP coding for influenza PR8 HA. Two weeks post-immunization the mice were mated.
- Non-immunized males mated to non-immunized females served as controls (DMN; dad-mom non-immunized).
- Immunized males with unimmunized (DI; dad immunized) or immunized females (DMI; dad-mom immunized), and immunized females with unimmunized males (MI; mom immunized).
Offspring from 1st, 2nd, and 4th litters at eight-ten weeks of age were intranasally inoculated with a sublethal dose of PR8 influenza and weight monitored for 14 days.
B). Percent of body weight drop aftern influenza infection (upper), and the corresponding AUC changes (lower). The data are from one experiment using litters from 3 separate mating. The number of mice (female/male) from 1st litters used for influenza challenge was 6/7 (DMN), 8/6 (DI), 3/9 (MI) and,8/7 (DMI); for the 2nd litters 1/3 (DMN), 6/9 (DI), 7/5 (MI) and 6/2 (DMI); for the 4th litters 2/9 (DMN), 8/13 (DI), 9/7 (MI) and 7/8 (DMI).
- After an earlier administration of mRNA-LNPs, the antibody response to a vaccine was inhibited. This inhibition of adaptive immune responses was relatively long-lasting, with effects seen for at least 4 weeks, and starting to wane after 8 weeks. This is consistent with the observation that a delay of the second dose of an mRNA vaccine to COVID from 3 weeks to 3 months significantly improves the antibody response.
- The data support that the inhibition of the adaptive immune responses could be overcome with the use of adjuvants, including Th1 (AddaVax) and Th2 (Alum) adjuvants. Also application at another location is preferred, because inhibition more pronounces at ipsilateral site.
- Mechanisms of increased protection against influenza and higher susceptibility by non-specific RNA LNP not elucidated, but must be related to lasting changes in innate immunity
- The highly inflammatory properties of the mRNA-LNP platform might have induced the inherited changes, and it would be very important to determine whether any such immune inheritance may be observed in humans vaccinated with mRNA vaccines: Cross-generational protection of infants of parents vaccinated
with BCG has recently been suggested in epidemiological studies
mRNA vaccines are a phantastic tool in modern vaccinology, but gaining more insight on how to modulate the LNP formulation to avoid side effects and preserve the adjuvant activity is urgently needed.
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