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

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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.
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Article
Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions and
immunogenicity of PEG-containing Covid-19 vaccines
Gergely Tibor Kozma,1,2
Tamás Mészáros
,1,2 Petra Berényi,1,2ka Facskó,1,2
Zsófia Pat
,3
Csaba Zs. Oláh
,4 Adrienne Nagy,5 Tamás Gyula Fülöp,6 Kathryn Anne Glatter,7
Tamás
Radovits,8 Béla Merkely,8
* János Szebeni
1,2,9*#
1 Nanomedicine Research and Education Center, Department of Translational Medicine,
Semmelweis University, Budapest, Hungary
2 SeroScience LCC, Budapest, Hungary
3 Department of Radiology, BAZ County Central Hospital and Borsod County University Teaching
Hospital and Miskolc University, Miskolc, Hungary
4 Department of Neurosurgery, BAZ County Central Hospital and Borsod County University
Teaching Hospital, Miskolc, Hungary
5 Department of Allergy, Heim Pál Children's Hospital, Budapest, Hungary
6 TECOdevelopment GmbH, Rheinbach, Germany
7 Gratz College, Philadelphia, Pennsylvania, USA
8 Heart and Vascular Center, Semmelweis University, Budapest, Hungary
9 Department of Nanobiotechnology and Regenerative Medicine, Faculty of Health Sciences,
Miskolc University, Miskolc, Hungary
*Equal contributors #Corresponding author
Abstract: 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
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 4, 2022. ; https://doi.org/10.1101/2022.10.03.22280227doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
2
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.
Key Words: Covid-19, SARS-CoV-2, mRNA, anaphylaxis, neutralizing antibodies, spike
protein, hypersensitivity reactions, ELISA, anti-PEG IgG, IgM
Correspondence: szebeni2@gmail.com, Szebeni.Janos@med.semmelweis-univ.hu
INTRODUCTION
The efficacy of mRNA-lipid nanoparticle (LNP)-based Covid-19 vaccines (Comirnaty and
Spikevax) in reducing death or severe illness from SARS-CoV-2 infections is well recognized.
However, as with all vaccinations, these vaccines may also have side effects in some people.
One of them is an allergic reaction, also known as a hypersensitivity reaction (HSR), whose most
severe manifestation is anaphylaxis.
Anaphylactic reactions to these vaccines, which are behind the mandated post-vaccination
observation of vaccinees, are very rare, and mostly controllable with epinephrine. Yet, there are
still life-threatening reactions and the phenomenon still represents a problem for severely allergic
people.1-18 Additionally, the specter of an allergic reaction helps fuel vaccine hesitancy.
Since the mRNA-LNP vaccines include polyethylene glycol (PEG) as an excipient, allergy
to PEG has been proposed as the mechanism of anaphylaxis. However, over time, it has been
shown that the overwhelming majority of these reactions are not IgE-mediated classic type I
allergies against PEG,1-17 leaving the underlying cause unclear. Among the studies addressing
this puzzle, the possible role of anti-PEG antibodies (Abs) was raised,1, 17, 19, 20 but conclusive
experimental or clinical evidence was not presented to date.
Most recently, two studies reported the rise of anti-PEG Abs in Spikevax-vaccinated
people,21, 22 implying specific binding of these Abs to PEG in the vaccine. In earlier studies,
anti-PEG Abs were shown to cause rapid loss of efficacy along with increased the risk of HSRs
to PEGylated urate oxidase,23, 24 and in another example, 3 anaphylactic reactions in patients
having very high pre-existing anti-PEG Abs led to early termination of a Phase II clinical trial
with the PEGylated RNA aptamer, Pegnivacogin.25, 26 These clinical data are consistent with
animal studies showing anti-PEG Ab-mediated anaphylaxis27 and accelerated blood clearance
(i.e., loss of efficacy) after i.v. injection of PEGylated liposomes.28, 29
Considering the parallelisms between the structures of mRNA-LNP vaccines and the above
reactogenic nanomedicines with potential waning of efficacy, we hypothesized that similarly to
these clinical and experimental examples, a contributing factor to the occasional anaphylactic
reactivity of mRNA-LNP vaccines and the individual variation of antiviral Ab levels they induce
could be associated with high levels of anti-PEG Abs.
Accordingly, we measured the plasma levels of anti-PEG IgG and IgM in allergic reactors
and nonreactors to Comirnaty and Spikevax vaccinations, using non-PEGylated Covid-19
vaccines as controls. These anti-PEG Abs were also correlated with the levels of SARS-CoV-2
neutralizing Abs, taken as endpoint for vaccine efficacy, and relating the postvaccination anti-
PEG Ab levels to the pre-vaccination values allowed us to investigate "bystander" anti-PEG
immunogenicity, similar to that reported for Spikevax.21, 22
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RESULTS AND DISCUSSION
Plasma anti-PEG IgG and IgM levels before vaccination: impacts of age, gender and
cosmetic use. Fig. 1 shows the anti-PEG IgG (A) and IgM (B) levels and distribution in the
blood of Covid-19 unvaccinated participants, and Table 1 lists the main statistical parameters
derived from these data. Contrary to the 10-76% range of anti-PEG Ab positivity in various
studies,21, 25, 26, 30-34 we found detectable levels of these Abs in 98-99% blood donors. Their
distribution was highly left-skewed, with considerable, 2-3 orders of magnitude span between the
lowest and highest values. The low, near baseline Ab levels in most samples is most easily
rationalized by low level immunization via the skin or per os by daily exposure to PEG, an
excipient in many oral, topical, and parenteral pharmaceutical products, hygiene and cosmetic
items, and processed food and beverages.35 It is also important to mention that Abs against PEG
and polysorbate 80 (PS-80), which is a branched PEG derivative, can mutually cross-react,36-38
and PS-80 is also widespread as an ingredient in drugs, vaccines, vitamins, food and drinks.35, 39
Thus, the anti-PEG ELISA does not necessarily distinguishes between these Abs.
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Figure 1. Pre-vaccination anti-PEG Antibody Concentrations in a Mixed Population of
Blood Donors. Anti-PEG-IgG (A, green) and anti-PEG-IgM (B, red) values were sorted in
growing order. The bottom bars (which look like lines) are absolute Ab concentrations.
The inserted probability distribution histograms are made from the log of absolute Ab
levels, grouped into bins on the abscissa.40 The normality of distribution was established
by the Shapiro-Wilk test. The anti-PEG Ab levels were determined as described in the
Methods, and the descriptive statistics of these data are shown in Table 1.
Table 1. Descriptive statistics of pre-vaccination anti-PEG-IgG and IgM levels and
distributions in humans
F/M ratios, female/male values; #, Samples below the detection limit were excluded from
normalized data analysis; *, significant difference between female and male at P<0.05 (in
framed values). Italicized values larger in female than in male by the F/M factor. CI,
confidence interval; m, mean; s, SD of log-normal distributions. The difference in male
and female number of subjects is due to 2 erroneous IgG determinations. Statistical
analysis was done by unpaired t test on logarithmic transformed data after their normal
distribution was checked by Shapiro-Wilk test.
Table 1 also shows that the prevalence and absolute levels of pre-vaccination anti-PEG IgM
was significantly higher in females compared to males. Questioning about cosmetic use revealed
that 1/3 of man were frequent cosmetic users versus >3/4 of women (Fig. 2A), and there was a
trend for higher IgG (Fig 2B), and significantly higher anti-PEG IgM in women (Fig 2C).
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Figure 2. Use of cosmetics by women and men and blood anti-PEG antibody levels in the
two genders. A) Ratio of frequent cosmetic users vs. no users or rare users in men and
women. B) Blood levels of anti-PEG-IgG (B) and anti-PEG-IgM (C) in frequent vs. no/rare
cosmetic users before COVID-19 vaccination. The anti-PEG Ab levels were determined as
described in the Methods. *P< 0.05 by 2-tailed t-test of logarithmic transformed data.
Correlating the anti-PEG IgG or IgM levels with age showed no significant correlation
(data not shown), and surprisingly, we found relatively high anti-PEG IgG (36 µg(eq)/mL) and
IgM (401 ng(eq)/mL) even in a baby less than one year old. Eight children in the 6-11 year-old
range also displayed high values (median of anti-PEG IgG and IgM were 27 µg(eq)/mL and 56
ng(eq)/mL, respectively).
We have also analyzed the relative changes of anti-PEG-IgG and IgM levels in the blood of 6
unvaccinated people in whom 2 measurements were available with different time intervals.
There was substantial variation of anti-PEG IgM over time, while the anti-PEG IgG remained
relatively constant in the same individuals (Supplement Fig S1-A). Although the n is too small
for making conclusions from these data, they raise the possibility of differential IgM and IgG
responses to transient PEG exposures that could take place in-between the blood withdrawals.
Accordingly, there was no significant correlation between the anti-PEG IgM and IgG levels
before vaccination (Fig. 1S-B). Nevertheless, we observed in 4 participants with a history of
allergy (red squares) and 3 with mastocytosis (green triangles) maximal anti-PEG-IgM levels
together with minimal anti-PEG IgG, or the opposite (Fig. 1S-B). These may be reflections of
immune abnormalities in these subjects calling for further attention to the cause and
consequences of the phenomenon. Our data also suggested higher anti-PEG IgG levels in those
donors who suffered from persistent allergies due to dust mite, mold, cat hair, foods, numerous
pollen type, chemicals, drugs, or cosmetics (data not shown).
Commenting on the higher prevalence of anti-PEG Ab positivity in our study relative to the
literature,21, 25, 26, 30-34 the exact reason is not clear. The discrepancy may partly be due to the
diverse quantitation methods for anti-PEG Abs in these studies,36 and partly to the different
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inclusion criteria of blood donors. For example, each referred reports on the prevalence of anti-
PEG Abs in the healthy population or drug-treated patients21, 25, 26, 30-34 used different antigens
and/or differing test conditions in the anti-PEG Ab ELISA assays. As for the inclusion criteria
of blood donors, we had a mixed population "enriched" with sporadic and chronic allergic people
and patients with mastocytosis.
The highly left-skewed distribution of pre-vaccination anti-PEG Abs in our study resembles
the distribution of these Abs in patients treated with the PEGylated RNA aptamer,
Pegnivacogin.25 On the other hand, our data may be the first to document such distribution in
PEG-medication-free subjects. The finding of increased prevalence of anti-PEG Abs in females
compared to males, a likely consequence of increased cosmetics use by women, is in line with
many studies reporting such information.21, 25, 26, 30-33
Anti-PEG IgG and IgM levels following vaccination with mRNA-LNP vaccines. Fig. 3A
and B show the individual and median anti-PEG IgG and IgM levels after vaccination with
mRNA vaccines as well as other, PEG-free Covid-19 vaccines (Sinopharm, Sputnik V and Astra
Zeneca), used for controlling the effect of PEG. The pre-vaccination baseline, also shown in Fig.
1, displayed substantial individual variation for both Ab subclasses, relative to which highly
significant rises after the first and 2 booster vaccinations were found only for Spikevax (Fig. 3A
and B). For Comirnaty, we found significant post-vaccination increase only in anti-PEG IgM
after the second jab (Fig. 3B). However, the considerable individual variation of anti-PEG Ab
levels over a broad range both before and after vaccination, and the irrelevance of vaccination
numbers when the question is the presence or absence of immunogenicity, led us to test another
approach of analysis, relating the maximal postvaccination values to the pre-vaccination
baselines for each vaccinees, regardless of injection number. This approach yielded highly
significant rises of both anti-PEG IgG and IgM for Comirnaty, also (Fig 3C). As for other
vaccines, the small number of preimmunization data did not yield statistically analyzable pre-
and post-vaccination pairs.
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Figure 3. Pre- and postvaccination anti-PEG Abs. Anti-PEG IgG (A, green rectangles)
and anti-PEG IgM (B, red circles) levels in people vaccinated with different Covid-19
vaccines shown on the X axis, wherein the numbers (1, 2 and 3) represent the order of
vaccinations. C) Relative rises of maximal postvaccination anti-PEG IgG and IgM levels
relative to the respective preimmunization value in people undergoing serial (at least 2)
immunizations with Comirnaty. Both absolute (A and B) and relative values (C) are
presented on log scale. Of note, the time elapsed between the pre- and postvaccination
measurements differ for each point. *, P< 0.05, ***, P< 0.001, ****, P<0.0001 by one-way
ANOVA of logarithmic transformed data followed by Dunnett test (A and B) and Wilcoxon
signed rank test of normalized data (C).
Our finding that Spikevax induces highly significant rise of anti-PEG Abs is consistent with
the mentioned recent data of Carreno et al.,22 and Ju et al,21 both groups coming to the same
conclusion. Regarding our finding that Comirnaty, too, causes similar, although less expressed
effect, which is seen only upon pairwise comparison, is also consistent with the latter two studies
although both concluded that there was no such effect in case of Comirnaty.21, 22 However, a
closer look at their data reveals very similar increases of anti-PEG IgG and IgM after vaccination
with Comirnaty as we have seen in a pairwise comparison (Fig 3C). Notably, in the study of
Carreno et al.22 Fig 2 shows 50-300% rise of prime/baseline anti-PEG IgG in 4/10 subjects,
expressed as log AUC, and Fig S1 in the study of Ju et al.21 shows 13/17, 4/9 and 10/15 endpoint
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dilution value pairs in cohorts 1, 2 and 3, respectively, where the post-booster values visibly
exceed the prevax values by > 20% up to several-fold on a log scale. These consistencies in
experimental data from 3 research groups despite the fact all used different antigens in their
ELISA (40 kDa-PEG,21 and 20kDa mPEG-BSA and 3.35-kDa free PEG22) provide strong
indication for the occasional presence of a weak bystander anti-PEG immunogenicity of these
vaccines. On the other end of the spectrum, the very high, outlier levels in a few anti-PEG Ab
"supercarriers" can be rationalized with induced Abs whose extensive formation may involve
positive feedback acceleration, a vicious cycle among anti-PEG Ab formation, C activation and
HSRs.41
Adverse reactions to Covid-19 mRNA-LNP vaccines. Table 2 categorizes all adverse
events described by the 195 Comirnaty or Spikevax recipients of this study. Based on the
definitions (see Table legend) we used for grouping the study participants according to reaction
type, clinical grades and Brighton levels,42 72% of vaccinees were reaction free (Grade 0), 20%
gave account of usual vaccine side effects (Grade 1), and 8% reported Grade 2 or 3 HSR
symptoms. There were more women among the reactors than man, and the 3 anaphylaxis cases
were all women.
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Table 2. Adverse reactions to mRNA/LNP Covid-19 vaccines.
Clinically-based
grading* Symptom types Brighton
Levels** Female Male All***
Grade 0 none NA 74 67 141 (72%)
Grade 1 usual vaccine
reactions 28 11 39 (20%)
Grade 2 hypersensitivity
reactions 2-3 7 5 12 (6 %)
Grade 3 1 3 0 3 (2 %)
Total 112 83 195
*Grade 0 is no adverse effect, Grade 1 "usual vaccine symptoms" include fever, arm pain,
local redness, weakness, headache, chills, pruritus, depression, abdominal pain, indigestion,
bloating, arthralgia, muscle pain, or fatigue. Grade 2 reactions represent allergic
symptoms which pass spontaneously with or without epinephrine, antihistamines, or other
pharmaceutical interventions. Grade 3 defines anaphylaxis or life-threatening reactions
requiring emergency care (resuscitation and/or hospitalization). Grade 4, death, N/A in
our study.
**Level of diagnostic certainty for anaphylaxis, as defined in the Brighton Collaboration's
anaphylaxis case definition guidelines.42 According to this system level 1 "true
anaphylaxis" is distinguished from other manifestations of allergy by its "diagnostic
certainty", defined by a complex matrix of major and minor criteria. Our Grade 3
reactions roughly correspond to Brighton level 1, i.e., true anaphylaxis, wherein at least a
major hemodynamic/circulatory or cardiopulmonary symptom (heart/back/limb pain,
hypo- or hypertension, (angio)edema, swelling of the lips, tongue or face) and a major skin
alteration (i.e., flushing, rash, erythema) are concurrently present. Brighton levels 2 and 3
(Grade 2) involve all other, non-life-threatening symptoms.
***The % values represent prevalence in the 195 participants of this study involving both
healthy and allergic people who were recalled for blood donation because of his/her HSR.
Thus, the data do not represent classical epidemiological surveillance of vaccine adverse
effects.
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Hypersensitivity reactions to mRNA-LNP vaccines and their association with anti-PEG
antibodies. The symptoms of 15 allergy responders included in this study are listed in Table 3,
along with the gender, age range, the vaccine type and number, and the severity of HSRs.
Table 3. Hypersensitivity reactions in recipients of mRNA-containing Covid-19 vaccines*
N
o Sex
#No
Age
range
(years
)
Vaccin
e
Jab
No
.
Clinic
al
Grade
Brighto
n
Level Symptoms
1 F #1 30-40 CMT 1 3 1 RAS, DYSP, LBP, ANA
2 F #2 50-60 CMT 4 ANA, ICH, WEA
3 F #3 50-60 CMT 1 ANA, HBP, IHR, SWTHR
4 F #4 30-40 SPV 1
2 2-3
CHL, MPAI, EXN, RAS, URT
5 F #5 20-30 CMT 2 HACH, NAUS, VOM, WEA, DEP, FEV,
RAS
6 F #6 40-50 CMT 2 ICH, BSE
7 F #7 40-50 CMT 1 RAS, ERY
8 F #8 20-30 CMT 3 RAS
9 F #9 60-70 CMT 3 FEV, HACH, ES
1
0 F #10 70-80 CMT 3 WEA, FEV, ERY
1
1 M #1 30-40 SPV 1 EXN, ERY, SWJ, TH, TON, LN, HR,
THE, THGL, DUR
1
2 M #2 30-40 CMT 1 RAS
1
3 M #3 30-40 SPV 3 RAS, ERY
1
4 M #4 20-30 CMT 2 ES
1
5 M #5 70-80 SPV 3 RAS, HOT, DGR
*Immediate and delayed reactions combined. Shaded area: "true" anaphylaxis reactions.
Vaccines: CMT, Comirnaty; SPV, Spikevax. Jab No., No of vaccination entailing HSR. ANA,
anaphylaxis; BSE, bloodshot eyes; CHL, chills; DEP, depression; DGR, dermatographia; DSP,
dyspnea; DUR, dark urine; ECZ, eczema; ERY, erythema; EXN, exanthema; ES, extrasystolia;
FEV, fever; HACH, headache; HBP, hypertension; HRS, hoarsenes; HOT, hotness; LBP,
hypotension; ICH, itching; MPAI, muscle pain; NCO, nasal congestion; NAU, nausea; RAS,
rashes; STR, stridor; SWA, SWT, SWJ, SWLN, SWE, SWTHR, SWTHGL, SWTON, swelling
of the arm, thenars, joints, lymph nodes, eyes, throat, thyroid gland, tonsils; IHR, tachycardia;
URT, urticaria; VOM, vomiting; WEA, weakness.
The above information in Table 3 reveals substantial individual variation of symptoms
afflicting mainly the circulatory, cardio-pulmonary, the nerve systems and the skin. Figure 4
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11
shows the anti-PEG IgG (A) and IgM (B) levels in reactor people displaying HSRs (R) compared
to non-rector (NR) subjects. Both Ab levels were significantly higher in the R group compared
to the NR, which suggests increased proneness of R people for anti-PEG Ab responses.
However, because the blood withdrawals were not coincidental with the HSRs, temporal linkage
between anti-PEG Ab rises and HSRs could not be assessed from these data.
Figure 4. Anti-PEG IgG (A), IgM (B) in reactor people displaying HSRs (R) compared to
non-rector (NR) subjects. The symptoms observed in the NR (no adverse effects and
Grade 1) and R (Grade 2 and 3) patients are detailed in Table 3. The time span between
Ab determinations and vaccinations was 4 months. **P< 0.01 by t-Test of logarithmic
transformed data.
SARS-CoV-2 neutralization antibody levels after multiple vaccinations: biphasic
dependence on anti-PEG Ab levels. Serial measurements of anti-SARS-CoV-2 neutralizing, S
protein-binding Abs (anti-S) in the plasma from 191 blood donors (n=361 tests) before and after
vaccinations multiple times (in the 1-9 range) showed several versions of Ab responses,
including small and large increases followed by steeper or weaker declines, constant low or high
levels, or strong or weak initial or late responses (Supplement Table S1). This individual
variation of antiviral immune response is in keeping with the variable and relatively short
duration of immunity provided by the current vaccines, necessitation booster injections.
Plotting the anti-S levels versus the anti-PEG IgG and IgM after the first and second
injections (Fig. 5A-D) in different subjects within 100 days after vaccination with Comirnaty
(without any sign of Covid-19 infection) suggested bell-shaped curves, which could arise, among
others, from a dose-dependent, biphasic correlation between anti-PEG Ab levels and anti-S
immunogenicity. This phenomenon was best seen in the case of anti-PEG IgM after the second
Comirnaty injection (Fig 5D).
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Figure 5. Anti-S Ab levels as a function of anti-PEG Abs within 100 days after the first or
the second vaccination with Comirnaty. Donors with any sign of Covid-19 infection were
excluded. Each points represent the highest Anti-S value obtained in serial plasma samples
in each individual. The goodness of lognormal Gaussian curve fitting is represented by R2
values on the figures.
The paralleling increases in both Abs on the left-side of the bell curve may reflect increasing
immune response against both PEG and the S-protein, while the waning levels of anti-S at higher
anti-PEG levels may reflect interference by the binding of anti-PEG Abs to the vaccine NPs and
possibly damage them. The latter assumption is based on the studies showing that the binding of
anti-PEG Abs, particularly IgM, to PEGylated liposomes can cause complement activation with
bilayer damage41, 43 as well as accelerated blood clearance of vesicles.27-29 Thus, anti-PEG Abs
may represent an important biological variable which critically impacts the efficacy of mRNA-
LNP vaccines in both directions.
OUTLOOK
There is consensus in the literature that the incidence of HSRs and anaphylaxis caused by
mRNA-LNP vaccinations is increased relative to that of traditional vaccines.1-17 Taken the ~1.3
anaphylaxis/million out of >25 million recipients of flu vaccines,44 as reference, the extent of
increase is in the 3-400-fold range (STable 2). Nevertheless, this potentially lethal side effect is
still considered as very rare, and its occurrence is further decreasing with time, as precautions are
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13
increased.18 However, the >1 billion mRNA-LNP injections given worldwide45 places the sheer
number of anaphylaxis cases probably in the tens of thousands (Table S2) and anaphylaxis is
only the tip of the iceberg, a small section in a broad spectrum of HSR symptoms. These HSRs,
even if mostly manageable, still represent a major acute health problem particularly for people
with severe allergies, among whom the incidence of Comirnaty-indued anaphylaxis is 0.7%
(STable 2), i.e., roughly 200-fold higher than in the normal population.11
We found in this study significantly increased levels of anti-PEG Abs in allergic reactor
subjects compared to non-reactors, which finding, by itself, does not prove causal relationship
between anti-PEG-Abs and HSRs. However, taken together with the unambiguous evidence for
at least a contributing role of anti-PEG Abs to anaphylaxis, and also efficacy loss, in the
discussed previous clinical and experiential studies with PEGylated NPs bearing similarities to
mRNA-LNP,23-29 the warning by Ganson et al. "we advise testing for pre-existing anti-PEG
antibodies during clinical trials of new PEGylated therapeutic agents"25 seems to be valid for
mRNA-LNP vaccinations, too, particularly in light of the need for frequent booster injections.
The potential anti-PEG bystander immunogenicity of these vaccines,21, 22 for which our study
also provided evidence, represents another reason for attention to these Abs in the light of
growing use of intrinsically reactogenic PEGylated nano-biopharmaceuticals. On the side of
welcome news, it is likely that in the non-allergic population only anti-PEG Ab supercarrier
people with very high levels of such Abs are at increased risk for HSRs and/or reduced efficacy
of PEGylated vaccines or drugs.
MATERIALS AND METHODS
Materials. The Covid-19 vaccines administered to study participants and used in our in vitro
studies were original, unexpired clinical batches. Their handling for storage and thawing was per
the manufacturer’s recommendations. The ELISA kits for measuring SARS-CoV-2
neutralization antibody (TE 1076) were from TECOMedical AG, Sissach, Switzerland.
Dulbecco’s phosphate-buffered saline (PBS) without Ca++/Mg++ and bovine calf serum, and
biotin-labeled goat polyclonal anti-porcine IgM were from Sigma Chemical Co. (St. Louis, MO,
USA).
Procedures involving Humans. The volunteers for blood sample donations were between
8 months and 85 years of age and were vaccinated once or several times with combinations of
mRNA (Comirnaty or Spikevax) and/or other vaccines, including Astra Zeneca, Jansen, Sputnik,
and Sinopharm. A portion of subjects had immune disorders, i.e., allergies and mastocytosis.
Blood was collected into EDTA vacutainers for plasma and clot activator tubes for serum. These
were stored in aliquots at -80 oC until the various assays were performed. We also obtained
samples from allergic reactors in other health care facilities, transported on dry ice. All
participants, parents of minors, filled out a consent form and questionnaire asking about their
age, medical and vaccination history, cosmetic use, and symptoms of HSRs, if they had
experienced them after Covid-19 vaccination. The study was approved by the Scientific and
Research Ethics Committee of the Hungarian Medical Research Council (52685-6/2022/EÜIG)
and, for Miskolc University, BORS-02/2021.
Antibody Tests. SARS-CoV-2 neutralization Abs specific against the receptor binding
region of viral spike protein (anti-S) were measured as described in Refs. 46, 47 using a kit
provided by TECOmedical AG, (Sissach, Switzerland, Catalog No. TE 1076). Serial
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14
measurements of blood anti-PEG IgM and IgG levels were performed with ELISA as described
earlier.27, 36, 48
Statistical Analysis. The applied approaches, specified in the figure legends, included
descriptive statistics, 2-tailed t-test on logarithmic transformed data, checking the normality of
distribution by Shapiro-Wilk test, one-way ANOVA followed by Dunnett test, Wilcoxon signed
rank test of normalized data and correlation analysis to test linear relationship between two
variables. ACKNOWLEDGMENTS
The authors are grateful to Marieluise Wippermann (TECOMedical, Sissach, Switzerland) for
providing the SARS-CoV-2 neutralization kits. Thanks are due to Dr. Judit Varkonyi for
recruiting patients with mastocytosis.
FUNDING
The financial support by the European Union Horizon 2020 projects 825828 (Expert) and
952520 (Biosafety) are acknowledged. This project was supported by a grant from the National
Research, Development, and Innovation Office (NKFIH) of Hungary (2020-1.1.6-JÖV
Ő
-2021-
00013). JS thanks the logistic support by the Applied Materials and Nanotechnology, Center of
Excellence, Miskolc University, Miskolc, Hungary.
CONFLICT OF INTEREST
The authors affiliated with SeroScience LLC are involved in the company’s contract research
service activity providing studies that were applied here. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript,
or in the decision to publish the results.
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SUPPLEMENTARY INFORMATION
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19
Figure S1. Anti-PEG Ab levels in peopl,e before Covid-19 vaccinations. A) Changes over
time in 6 subjects who gave blood 2 times before vaccination against Covid-19. The
second/first anti-PEG IgG and IgM ratios were plotted against the time elapsed time
between the 1st and 2nd blood withdrawal. B) Lack of correlation of plasma anti-PEG-
IgM and anti-PEG-IgG in all blood donors before vacination (black dots), except in those
with allergy (red squares) or mastocytosis (green triangles), in whom maximal levels of
anti-PEG IgG were associated with minimal values of anti-PEG IgM. Antibody levels were
determined as described in the Methods.
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20
Table S1. Inter- and intra-individual variation of anti-S levels in Covid-19 vaccine
recipients*
No** Test (repeat) No
1 2 3 4 5 6 7 8 9 n***
IU/mL
1 1031 276
2
2 315 154 109 1421 1284 >500
6
3 >500
1
4 >500
1
5 182 956 309 >500
4
6 0 69 33 >500
4
7 16 48 126 38 120 168
6
8 0 0 0 94 68 45 34 340 276 9
9 70
1
10 211
1
11 67 1995 1266 498 206 135 91
7
12 >500
1
13 0 0 308
3
14 0 0 1792 1082 68 34
6
15 3991
1
16 0
1
17 0 17 1257 >500
4
18 11592 >500
2
19 0 99
2
20 0 18 19 357 353 97 31 1380 >500 9
21 0
1
22 2436 688 >500 2782
4
23 122 966 >500
3
24 0 38 20 663 208
5
25 38 5495 2537 748 398 313 >500
7
26 10 36 141
3
27 171 2054 1379 359 407 375 >500
7
28 162 14
2
29 440 486
2
30 402
1
31 19 16 431 513 496 1349 >500
7
32 13 330
2
33 24 930
2
34 >500
0
35 1350 687 1214 1778 1263 735 >500 >500
8
36 11 13 704
3
37 0 6 72
3
38 0 79 1478 1458 1243 2279
6
39 65 194 109
3
40 388
1
41 23 2086
2
42 36
1
43 1064 4406 >500
3
44 29 989 478
3
45 255 813
2
46 419 1939 >500
3
47 671 5713 >500
3
48 113
1
49 52
1
50 15 67
2
51 403 19 948 95
4
52 1016 359 450 >500
4
53 1812 2495 1123 >500
4
54 51
1
55 681
1
56 51 25 160
3
57 13
1
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 4, 2022. ; https://doi.org/10.1101/2022.10.03.22280227doi: medRxiv preprint
21
58 294 0 0
3
59 >500 0 0
2
60 358
1
61 8
1
62 8
1
63 20 1457
2
64 7
1
65 2490 2262 >500
3
66 611 330
2
67 18 54 4540 1927
4
68 82
1
69 100
1
70 2211
1
71 6
1
72 7
1
73 8
1
74 13
1
75 30
1
76 88 63 32 2893 >500 >500
6
77 2021
1
78 1084
1
79 371
1
80 362
1
81 3013
1
82 30
1
83 339 81
2
84 169
1
85 318
1
86 9
1
87 21 9 1255
3
88 465 129 3019
3
89 12 5887 >500
3
90 420
1
91 362
1
92 33 2203 >500
3
93 118 48
2
94 21 0
2
95 328 409 190
3
96 406
1
97 1303 >500
2
98 6747 >500
2
99 890
1
100 46
1
101 1601
1
102 8
1
103 61
1
104 3749
1
105 6352 >500
2
106 6 78 69
3
107 14 660
2
108 310 444
2
109 64
1
110 5
1
111 2959 >500
2
112 409
1
113 229
1
114 413
1
115 301 4331
2
116 85
1
117 272
1
118 401
1
119 4990
1
120 1480
1
121 1337
1
122 542
1
123 442 407
2
124 44
1
125 21
1
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 4, 2022. ; https://doi.org/10.1101/2022.10.03.22280227doi: medRxiv preprint
22
126 889 >500
2
127 640
1
128 1554
1
129 8
1
130 464
1
131 193
1
132 42 1484
2
133 9
1
134 1443
1
135 538
1
136 255 378 117 228 145
5
137 52
1
138 892
1
139 0
1
140 0
1
141 0
1
142 1043
1
143 1634
1
144 542
1
145 143
1
146 432
1
147 69
1
148 21
1
149 719
1
150 204
1
151 135
1
152 577
1
153 1016
1
154 10041 >500
2
155 253
1
156 22
1
157 123
1
158 6633
1
159 6241
1
160 156
1
161 91
1
162 72
1
163 >500 >500
2
164 375
1
165 548
1
166 425
1
167 >500 >500
2
168 >500
1
169 235
1
170 >500
1
171 >500
1
172 >500
1
173 >500
1
174 12
1
175 287
1
176 >500
1
177 >500
1
178 >500
1
179 >500
1
180 >500
1
181 >500
1
182 >500
1
183 >500
1
184 32
1
185 93
1
186 >500
1
187 >500
1
188 >500
1
189 >500
1
190 >500
1
191 418
1
all test N: 361
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 4, 2022. ; https://doi.org/10.1101/2022.10.03.22280227doi: medRxiv preprint
23
*, All data pooled, regardless of vaccine type or booster combinations, or time after the
injections. **Blood donor number (only in this Table for identification) ***, total no of serial
testing in a donor. >500 indicates undefined values above 500 IU/mL, the upper limit of
detection. In these samples finetuning of the values by further dilution of samples could not be
done.
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 4, 2022. ; https://doi.org/10.1101/2022.10.03.22280227doi: medRxiv preprint
24
Table S2. Different statistics on the incidence of anaphylaxis to mRNA-LNP vaccines
Vaccine Reaction n* Total n** Ana/million Ana % References
Comirnaty 47 9,943,247 5 0.00047
5, 49
Spikevax
19
7,581,429
3
0.00025
Comirnaty
46
14,475,979
3
0.00032
18
Comirnaty
3730
242,000,000
15
0.00154
48
Spikevax
1455
154,000,000
9
0.00094
Comirnaty
+ Spikevax 1 4000 250 0.02500
8
Comirnaty
7
25929
270
0.02700
9
Spikevax
9
38971
231
0.02309
Comirnaty
14
31635
443
0.04425
3
Spikevax
3
7260
413
0.04132
Comirnaty 3
429***
2,331
0.699
11
80,943**** 37 0.00371
*, Number of reported anaphylaxis or "severe" HSR cases; **, Total n of injections or vaccine
recipients; ***, Only highly allergic people were included in the statistic; ****, Normalized to
the healthy population based on the ratio of highly allergic people among people with any
allergic complain (5,3%)11 and a rough estimate of 10% prevalence of different allergies
worldwide (Wikipedia).
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 4, 2022. ; https://doi.org/10.1101/2022.10.03.22280227doi: medRxiv preprint
... 28 Regardless of C activation, the likely causal role of anti-PEG Abs in mRNA-LNP vaccineinduced HSRs/anaphylaxis was raised by many groups, 1,8,15,16,18,31 and we found significant correlation between the blood levels of anti-PEG Abs and rise of HSR/anaphylaxis in recipients of Comirnaty and Spikevax. 32 Nevertheless, all above facts provide only indirect proofs of a causal role of anti-PEG Abs in Comirnaty-induced HSRs; conclusive experimental or clinical evidence is still lacking. Accordingly, the goal of the present study was to obtain such evidence using the anti-PEG hyperimmune pig model, 33 which showed the acceleration of HSR to anaphylaxis to PEGylated liposomal doxorubicin (Doxil) if the blood level of anti-PEG IgM had been increased by prior vaccination with drug-free Doxil (Doxebo). ...
... The model applied in the present study has many unique benefits. One is that further sensitization of already sensitive pigs for CARPA provides a model for those people who have very high anti-PEG Ab levels (i.e., anti-PEG Ab supercarriers), 32 and, hence, are prone not only to vaccine-induced HSR/anaphylaxis but also to those caused by many other PEGylated drugs and household items. Yet another unique benefit is that the hemodynamic and cardiopulmonary changes mimic those human circulatory abnormalities (mainly cardiopulmonary distress, acute myocardial infarction), that make cardiac anaphylaxis life-threatening. ...
Preprint
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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.
... Furthermore, since both Vepesid and Comirnaty can rarely cause hypersensitivity (anaphylactic) reactions, 12-16 another concern for the patient was a potential increase in the risk for such reactions, because of Comirnaty-induced rise of anti-PEG antibody levels, a likely contributing factor to the anaphylaxis to this vaccine. [16][17][18][19] Thus, we have also measured anti-PEG IgM levels in the patient's blood, as well as neutrophil and lymphocyte counts, complement levels, and nonspecific cellular immune response to T cell stimulation after vaccinations, taken as measures of innate immune response to the vaccines under chemotherapy. ...
... In fact, the anti-PEG IgM measurements in our patient was done as part of a different project aimed to establish the risk of allergic people to develop anaphylactic reaction to Comirnaty, which contains PEG. 15,16 High blood levels of such antibodies have been implicated in the anaphylactic reactions to numerous PEGylated drugs, [17][18][19]33,34 Furthermore there is a suspicion that the increased occurrence of such reactions after mRNA vaccines 35-40 may be due, at least in part, to the presence of high levels of anti-PEG antibodies. 15,16,41 In fact, a recent study on 55 people receiving Comirnaty showed the common presence of anti-PEG IgG and IgM, which were boosted by vaccination by a mean of 2-3-fold, up to 13-17-fold in some individuals. ...
Article
Full-text available
The rising need for repeated booster vaccinations against SARS-CoV-2 infections raises the question of whether chronic immunosuppressive chemotherapies influence the efficacy of vaccination. Here, we present the case of a 70-year-old post-thymoma surgery patient who received Vepesid (etoposide, Xediton Pharmaceuticals Inc) chemotherapy for six months before vaccination with Comirnaty (Pfizer-BioNTech COVID-19 mRNA Vaccine). The first two vaccinations elicited only minimal increases of IgG antibodies specific against the receptor-binding domain (RBD) on the spike protein (S1), while the third vaccination was effective in providing high, slowly subsiding antibody titers over a 7-month period. The patient also developed a cellular immune response after the third vaccination. Also, measuring of anti-polyethylene glycol (PEG) IgM titers before and after vaccinations showed no immunogenicity for PEG. Later, a single dose of Sinopharm (China National Pharmaceutical Group) inactivated virus-type vaccine was administered, which also modestly increased the level of IgG. A symptomless COVID-19 infection, however, greatly increased the serum level of anti-RBD IgG, which later subsided. This case confirms that an effective immune response can be achieved with a series of COVID-19 vaccinations despite cytostatic treatment in an old thymus cancer surviving patient in the absence of adverse reactions.
Article
Despite the success of mRNA-based vaccines against infectious diseases (including COVID-19), safety concerns have been raised relating to the lipid nanoparticles (LNPs) used to deliver the mRNA cargo. Antibodies against the polyethylene glycol (PEG) coating on these non-viral vectors are present in the general population and can in some instances induce allergic reactions. Furthermore, treatment with PEGylated therapeutics may increase the plasma concentration of such anti-PEG antibodies. The widespread use of PEGylated nanoparticles for mRNA vaccines concerns researchers and clinicians about a potential rise in future cases of allergic reactions against mRNA vaccines and cross-reactions with other PEGylated therapeutics. To determine if vaccination with Comirnaty increased the plasma concentration of antibodies against LNPs, we investigated the blood plasma concentration of anti-LNP antibodies in healthy individuals before and after vaccination with the mRNA-based COVID-19 vaccine Comirnaty (BNT162b2). Blood samples were acquired from 21 healthy adults before vaccination, 3-4 weeks after the first vaccination dose but before the second dose, and 2-6 months after the second (booster) dose. The blood plasma concentration of antibodies recognizing the LNPs was analyzed using a microscopy-based assay capable of measuring antibody-binding to individual authentic LNPs. No significant increase in anti-LNP antibodies was observed after two doses of Comirnaty. The LNPs used for intramuscular delivery of mRNA in the vaccine against COVID-19, Comirnaty, do, therefore, not seem to induce the generation of anti-vector antibodies.
Article
The successful mRNA vaccines against COVID-19 contain polyethylene glycol (PEG) to stabilize the lipid nanoparticles. Recent data show that PEG-specific antibodies can be induced or boosted by mRNA vaccination. Further research is needed to study the potential links between PEG-specific antibodies, vaccine reactogenicity and enhanced clearance of other PEG-containing medicines.
Article
Full-text available
Background In December 2020 the Israeli Ministry of Health (IMoH) launched a national vaccination campaign against SARS-CoV-2. Concomitant sporadic reports on anaphylactic responses in other countries raised safety concerns at the outset of this operation. Objective To characterize reports on allergic reactions to COVID-19 vaccines. Methods Allergy events were reported by health care professionals throughout the country to IMoH Division of Epidemiology via a web-based computerized national vaccine registry. The study period was from December 19, 2020 to September 13, 2021 during which 14,475,979 injections were administered. Results Allergic reactions were reported in 463 subjects, of whom 99.3% received Pfizer-BioNTech BNTT162B2. Reporting rate was 106 per million in December 2020. From January to May 2021 a reduction was observed to 66, 18, 14, 8 and 0 per million, and reporting remained low until September. Subject mean age was 48.9 ± 16.7 years (range 15-96) with a female preponderance of 78%. Epinephrine was administered in 34 subjects. Validated immediate allergy was observed in only 37 cases (8%) suggesting 2.5-3.3 bona fide reactions per million. In subjects with reactions that were classified as severe (n=47) plausible allergy was identified in 36-41% of cases. A history of allergy was associated with high false reporting of immediate reactions (83%). Allergic events following the first dose did not compromise adherence to subsequent doses. Conclusion Excessive reporting of allergy declined over time and did not affect adherence to vaccination. The existence of previous allergy may impact reporting profiles but not the occurrence of vaccine allergy.
Article
Full-text available
Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG, but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle-immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0-70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68-16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9-377.1) and 2.64-fold (0.76-12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. 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. PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle-leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA vaccines should be monitored. It may be useful to identify suitable alternatives to PEG for developing next-generation LNP vaccines to overcome PEG immunogenicity in the future.
Article
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Conventional vaccines have been widely studied, along with their risk of causing allergic reactions. These generally consist of mild local reactions and only rarely severe anaphylaxis. Although all the current COVID-19 vaccines marketed in Europe have been shown to be safe overall in the general population, early post-marketing evidence has shown that mRNA-based vaccines using novel platforms (i.e., lipid nanoparticles) were associated with an increased risk of severe allergic reactions as compared to conventional vaccines. In this paper we performed an updated literature review on frequency, risk factors, and underlying mechanisms of COVID-19 vaccine-related allergies by searching MEDLINE and Google Scholar databases. We also conducted a qualitative search on VigiBase and EudraVigilance databases to identify reports of "Hypersensitivity" and "Anaphylactic reaction" potentially related to COVID-19 vaccines (Comirnaty, Spikevax, Vaxzevria and COVID-19 Janssen Vaccine), and in EudraVigilance to estimate the reporting rates of "Anaphylactic reaction" and "Anaphylactic shock" after COVID-19 vaccination in the European population. We also summarized the scientific societies' and regulatory agencies' recommendations for prevention and management of COVID-19 vaccine-related allergic reactions, especially in those with a history of allergy.
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A tiny fraction of people immunized with lipid nanoparticle (LNP)-enclosed mRNA (LNP-mRNA) vaccines develop allergic symptoms following their first or subsequent vaccinations, including anaphylaxis. These reactions resemble complement (C) activation-related pseudoallergy (CARPA) to i.v. administered liposomes, for which pigs provide a naturally oversensitive model. Using this model, we injected i.v. the human vaccination dose (HVD) of BNT162b2 (Comirnaty, CMT) or its 2-fold (2x) or 5-fold (5x) amounts and measured the hemodynamic changes and other parameters of CARPA. We observed in 6 of 14 pigs transient pulmonary hypertension along with thromboxane A2 release into the blood and other hemodynamic and blood cell changes, including hypertension, granulocytosis, lymphopenia, and thrombocytopenia. One pig injected with 5x CMT developed an anaphylactic shock requiring resuscitation, while a repeat dose failed to induce the reaction, implying tachyphylaxis. These typical CARPA symptoms could not be linked to animal age, sex, prior immune stimulation with zymosan, immunization of animals with Comirnaty i.v., or i.m. 2 weeks before the vaccine challenge, and anti-PEG IgM levels in Comirnaty-immunized pigs. Nevertheless, IgM binding to the whole vaccine, used as antigen in an ELISA, was significantly higher in reactive animals compared to non-reactive ones. Incubation of Comirnaty with pig serum in vitro showed significant elevations of C3a anaphylatoxin and sC5b-9, the C-terminal complex. These data raise the possibility that C activation plays a causal or contributing role in the rare HSRs to Comirnaty and other vaccines with similar side effects. Further studies are needed to uncover the factors controlling these vaccine reactions in pigs and to understand their translational value to humans.
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Although allergic responses to the mRNA COVID-19 vaccines are rare, recent reports have suggested that a small number of individuals with allergy to polyethylene glycol (PEG), a component of the mRNA lipid nanoshell, may be at increased risk of anaphylaxis following vaccination. In this report, we describe a case of a patient who received an mRNA COVID-19 vaccine, experienced anaphylaxis, and was subsequently confirmed to have anti-PEG allergy by skin prick testing. The patient had previously noticed urticaria after handling PEG powder for their occupation and had a history of severe allergic response to multiple other allergens. Importantly, as many as 70% of people possess detectable levels of anti-PEG antibodies, indicating that the detection of such antibodies does not imply high risk for an anaphylactic response to vaccination. However, in people with pre-existing anti-PEG antibodies, the administration of PEGylated liposomes may induce higher levels of antibodies, which may cause accelerated clearance of other PEGylated therapeutics a patient may be receiving. It is important to improve awareness of PEG allergy among patients and clinicians.
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Importance As of May 2021, more than 32 million cases of COVID-19 have been confirmed in the United States, resulting in more than 615 000 deaths. Anaphylactic reactions associated with the Food and Drug Administration (FDA)–authorized mRNA COVID-19 vaccines have been reported. Objective To characterize the immunologic mechanisms underlying allergic reactions to these vaccines. Design, Setting, and Participants This case series included 22 patients with suspected allergic reactions to mRNA COVID-19 vaccines between December 18, 2020, and January 27, 2021, at a large regional health care network. Participants were individuals who received at least 1 of the following International Statistical Classification of Diseases and Related Health Problems, Tenth Revision anaphylaxis codes: T78.2XXA, T80.52XA, T78.2XXD, or E949.9, with documentation of COVID-19 vaccination. Suspected allergy cases were identified and invited for follow-up allergy testing. Exposures FDA-authorized mRNA COVID-19 vaccines. Main Outcomes and Measures Allergic reactions were graded using standard definitions, including Brighton criteria. Skin prick testing was conducted to polyethylene glycol (PEG) and polysorbate 80 (P80). Histamine (1 mg/mL) and filtered saline (negative control) were used for internal validation. Basophil activation testing after stimulation for 30 minutes at 37 °C was also conducted. Concentrations of immunoglobulin (Ig) G and IgE antibodies to PEG were obtained to determine possible mechanisms. Results Of 22 patients (20 [91%] women; mean [SD] age, 40.9 [10.3] years; 15 [68%] with clinical allergy history), 17 (77%) met Brighton anaphylaxis criteria. All reactions fully resolved. Of patients who underwent skin prick tests, 0 of 11 tested positive to PEG, 0 of 11 tested positive to P80, and 1 of 10 (10%) tested positive to the same brand of mRNA vaccine used to vaccinate that individual. Among these same participants, 10 of 11 (91%) had positive basophil activation test results to PEG and 11 of 11 (100%) had positive basophil activation test results to their administered mRNA vaccine. No PEG IgE was detected; instead, PEG IgG was found in tested individuals who had an allergy to the vaccine. Conclusions and Relevance Based on this case series, women and those with a history of allergic reactions appear at have an elevated risk of mRNA vaccine allergy. Immunological testing suggests non–IgE-mediated immune responses to PEG may be responsible in most individuals.
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Polyethylene glycol (PEG) is a flexible, hydrophilic simple polymer that is physically attached to peptides, proteins, nucleic acids, liposomes, and nanoparticles to reduce renal clearance, block antibody and protein binding sites, and enhance the half-life and efficacy of therapeutic molecules. Some naïve individuals have pre-existing antibodies that can bind to PEG, and some PEG-modified compounds induce additional antibodies against PEG, which can adversely impact drug efficacy and safety. Here we provide a framework to better understand PEG immunogenicity and how antibodies against PEG affect pegylated drug and nanoparticles. Analysis of published studies reveals rules for predicting accelerated blood clearance of pegylated medicine and therapeutic liposomes. Experimental studies of anti-PEG antibody binding to different forms, sizes, and immobilization states of PEG are also provided. The widespread use of SARS-CoV-2 RNA vaccines that incorporate PEG in lipid nanoparticles make understanding possible effects of anti-PEG antibodies on pegylated medicines even more critical.
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Two messenger RNA (mRNA)-based vaccines are widely used globally to prevent coronavirus disease 2019 (COVID-19). Both vaccine formulations contain PEGylated lipids in their composition, in the form of polyethylene glycol [PEG] 2000 dimyristoyl glycerol for mRNA-1273, and 2 [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide for BNT162b2. It is known that some PEGylated drugs and products for human use which contain PEG are capable of eliciting immune responses that lead to to detectable PEG-specific antibodies in serum. In this study, we determined if any of the components of mRNA-1273 or BNT162b2 formulations elicited PEG-specific antibody responses in serum by enzyme linked immunosorbent assay (ELISA). We detected an increase in the reactivity to mRNA vaccine formulations in mRNA-1273 but not BNT162b2 vaccinees’ sera in a prime-boost dependent manner. Furthermore, we observed the same pattern of reactivity against irrelevant lipid nanoparticles from an influenza virus mRNA formulation and found that the reactivity of such antibodies correlated well with antibody levels against high and low molecular weight PEG. Using sera from participants selected based on the vaccine-associated side effects experienced after vaccination, including delayed onset, injection site or severe allergic reactions, we found no obvious association between PEG antibodies and adverse reactions. Overall, our data shows a differential induction of anti-PEG antibodies by mRNA-1273 and BNT162b2. The clinical relevance of PEG reactive antibodies induced by administration of the mRNA-1273 vaccine, and the potential interaction of these antibodies with other PEGylated drugs remains to be explored.
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After over a billion of vaccinations with messenger RNA-lipid nanoparticle (mRNA-LNP) based SARS-CoV-2 vaccines, anaphylaxis and other manifestations of hypersensitivity can be considered as very rare adverse events. Although current recommendations include avoiding a second dose in those with first-dose anaphylaxis, the underlying mechanisms are unknown; therefore, the risk of a future reaction cannot be predicted. Given how important new mRNA constructs will be to address the emergence of new viral variants and viruses, there is an urgent need for clinical approaches that would allow a safe repeated immunization of high-risk individuals and for reliable predictive tools of adverse reactions to mRNA vaccines. In many aspects, anaphylaxis symptoms experienced by the affected vaccine recipients resemble those of infusion reactions to nanomedicines. Here we share lessons learned over a decade of nanomedicine research and discuss the current knowledge about several factors that individually or collectively contribute to infusion reactions to nanomedicines. We aim to use this knowledge to inform the SARS-CoV-2 lipid-nanoparticle-based mRNA vaccine field.