key: cord-1006818-92ulcl62
authors: Carreno Quiroz, J. M.; Singh, G.; Tcheou, J.; Srivastava, K.; Gleason, C.; Muramatsu, H.; Desai, P.; Aberg, J. A.; Miller, R. L.; PARIS study group,; Pardi, N.; Simon, V.; Krammer, F.
title: mRNA-1273 but not BNT162b2 induces antibodies against polyethylene glycol (PEG) contained in mRNA-based vaccine formulations
date: 2022-04-17
journal: nan
DOI: 10.1101/2022.04.15.22273914
sha: 0dcae6c0c807f90bd0587eb79e3b57053f96afb8
doc_id: 1006818
cord_uid: 92ulcl62

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 that contain PEG, are capable of eliciting immune responses, leading 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.

The mRNA used as an irrelevant control was designed based on the influenza virus 154 B/Colorado/06/2017 neuraminidase (NA) sequence. Production of the mRNA was performed as 155 described earlier (16, 17) . Briefly, the codon-optimized NA gene was synthesized (Genscript) and 156 cloned into an mRNA production plasmid. A T7-driven in vitro transcription reaction (Megascript, 157 Ambion) using linearized plasmid template was performed to generate mRNA with 101 158 nucleotide long poly(A) tail. Capping of the mRNA was performed in concert with transcription 159 through addition of a trinucleotide cap1 analogue, CleanCap (TriLink) and m1Ψ-5'-triphosphate 160 (TriLink) was incorporated into the reaction instead of UTP. Cellulose-based purification of NA 161 mRNA was performed as described (18) . The mRNA was then tested on an agarose gel before 162 storing at -20°C. 163

The cellulose-purified m1Ψ-containing NA mRNA was encapsulated in LNPs using a 164 self-assembly process as previously described wherein an ethanolic lipid mixture of ionizable 165 cationic lipid, phosphatidylcholine, cholesterol and polyethylene glycol-lipid was rapidly mixed 166

with an aqueous solution containing mRNA at acidic pH (19) . The RNA-loaded particles were 167 characterized and subsequently stored at -80°C at a concentration of 1 mg/ml. 168 169 Expression and purification of recombinant SARS-CoV-2 spike protein. 2 spike protein was produced using a mammalian cell protein expression system. Briefly, the 171 spike (S) gene sequence (GenBank: MN908947) was cloned into a mammalian expression vector 172 pCAGGs, as described (20, 21) . Protein was expressed using the Expi293 Expression System 173 (Thermo Fisher Scientific), according to the manufacturer's instructions. Cell culture supernatant 174 was collected and clarified by centrifugation at 4000 x g, filtered, and purified with Ni-175 nitrilotriacetic acid (NTA) agarose (QIAGEN). The purified protein was concentrated using Amicon 176

Ultracell (Merck Millipore) centrifugation units, and the buffer was exchanged to a phosphate 177 buffer solution (PBS, pH 7.4). Proteins were stored at -80°C until use. 178 179 Enzyme linked immunosorbent assay (ELISA). Antibody titers in vaccinees' sera were 180 determined against the recombinant trimeric spike protein of wild type SARS-CoV-2 as previously 181 described (21, 22) . Spike and BSA ELISAs were performed using phosphate-buffered saline (PBS) 182 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. ;  7 with 0.1% Tween-20 (PBS-T; Fisher Scientific) in washing, blocking, and diluting solutions. mRNA 183 vaccines BNT162b2 (from Pfizer) and mRNA-1273 (from Moderna), irrelevant mRNA LNPs, multi-184 PEGylated bovine serum albumin (mPEG-BSA, 20 kDa, Life Diagnostics, Inc), and low molecular 185 weight PEG (3.35kDa, Sigma) based ELISAs were performed using a modified protocol in which 186

Tween-20 was excluded from washing/Ab solutions. Briefly, polystyrene 96-well microtiter plates 187 (Thermo Fisher Scientific) were coated overnight with mPEG-BSA (2μg/ml), BNT162b2 or mRNA-188 1273 vaccine formulations (25μl/10ml), irrelevant mRNA lipid nanoparticles (0.5μg/ml) or BSA 189 (1% solution, MP Biomedicals). For IgE and BSA controls (shown in Supplementary Figure 2) , ELISA 190 plates were coated with 2μg/ml of an IgE isotype control (Invitrogen) and BSA (1% solution, MP 191 Biomedicals) respectively. The following day, wells were washed and blocked with 200 ul of 3% 192 non-fat milk (AmericanBio) in PBS for 1 h at room temperature (RT). After 1 h incubation, blocking 193 solution was removed and pre-diluted sera (in PBS 1% non-fat milk) were added at an initial 194 dilution of 1:50 followed by 2-fold serial dilutions. After 2 h incubation, plates were washed three 195 times with PBS and then incubated for 1 hour with anti-human IgG (Fab-specific) horse radish 196 peroxidase (HRP) secondary antibody produced in goat (Sigma-Aldrich), or IgM-HRP (Southern 197 Biotech) at a 1:3000 in 1% milk PBS. Specific spike/PEG-IgE antibodies were assessed by 198 incubating with an anti-IgE HRP conjugated antibody (Invitrogen) for 1 h at a 1:2000 dilution. For 199 the IgE control, plates were incubated with serial dilutions (2-fold) of the anti-IgE HRP conjugated 200 antibody (Invitrogen) starting at a 1:1000 dilution for 1 h. For the BSA control, plates were 201 incubated with serial dilutions (2-fold) of an anti-albumin (bovine serum) rabbit IgG fraction (anti-202 BSA, Invitrogen) starting at a 1:1000 dilution for 1 h, followed by three washes with PBS and 203 addition of a donkey anti-rabbit IgG HRP conjugated antibody (CiteAb) at a 1:1000 dilution. Plates 204 were washed three times with PBS and 100μl/well of O-phenylenediamine dihydrochloride (OPD) 205 substrate (SigmaFast OPD; Sigma-Aldrich) were added. After 10 min incubation at RT, the 206 reaction was stopped by addition of 50 μl of 3 M HCl solution. The optical density (OD) was 207 measured at 490 nm using a Synergy 4 plate reader (BioTek). Data were captured in excel and 208 are urea under the curve (AUC) values were determined using Prism 9 (GraphPad Software, San 209

Diego, CA, USA). 210 . CC-BY-NC-ND 4.0 International license It 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 April 17, 2022. ;  8 Statistical analyses. Data plotting and statistical analyses were performed using GraphPad Prism 211 9 (GraphPad Software, San Diego, CA, USA). Statistically significant differences between post-212 prime/post-boost vs baseline antibody levels were measured using a one-way ANOVA with 213

Tukey's multiple comparisons test. All adjusted P values of <0.05 were considered statistically 214 significant with a confidence interval of 95%. PBS and assessed binding of IgG antibodies. We found that sera collected after vaccination with 231 mRNA-1273 had increasing reactivity against both the BNT162b2 (Fig. 1C ) and mRNA-1273 ( Fig.  232   1D ) formulations, and that the increased reactivity was vaccination-dependent, with a moderate 233 increase after the prime administration and higher levels induced after the boost. Interestingly, 234 this increase in reactivity was not evident in sera from individuals receiving the BNT162b2 235 vaccine, either against the BNT162b2 or mRNA-1273 formulations (Figs. 1E and 1F) . Overall, these 236 data suggest that mRNA-1273 but not BNT162b2 vaccination induces antibodies against some 237 component(s) of the vaccine formulations. 238 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. ;  9 Antibodies reactive towards the vaccine formulation are directed against the lipid 239 nanoparticles and react with polyethylene glycol (PEG). Lipid nanoparticles contained in the 240 currently used mRNA vaccine formulations, as well as other drugs and other cosmetic and health 241 products for human use, contain PEG and have the potential to elicit immune responses against 242 it (10, 13-15). To dissect the target within the vaccine formulation to which mRNA-1273-induced 243 antibodies bind, we coated ELISA plates with LNPs carrying a SARS-CoV-2 unrelated, irrelevant 244 mRNA (encoding influenza virus neuraminidase). Similar to the reactivity overserved against the 245 BNT162b2 and mRNA-1273 formulations, we detected an increase in the reactivity against the 246 irrelevant mRNA-LNPs in individuals receiving the mRNA-1273 vaccine (Fig. 2B) , but not in those 247 ones vaccinated with BNT162b2 ( Fig. 2A) , suggesting the reactivity is independent of the 248 sequence of the mRNA contained in the formulation. 249 250 Next, we assessed whether vaccination induced antibodies reacted to PEG. We measured the 251 binding of sera from mRNA-1273 and BNT162b2 vaccinated individuals to a PEGylated form of 252 BSA (PEG-BSA) containing high molecular weight PEG (20kDa). Again, we found that individuals 253 vaccinated with mRNA-1273, had an increase in antibodies against PEG-BSA in a vaccination-254 dependent manner (Fig. 2D) , whereas no significant increase of antibody titers in individuals 255 receiving the BNT162b2 vaccine was observed (Fig. 2C) . As an alternative experimental approach, 256 we coated high binding polystyrene plates directly with a low molecular weight PEG (3.35 kDa) 257 molecule and performed similar ELISAs. Although this method seemed to be less sensitive than 258 the PEG-BSA ELISA, similarly we observed that individuals vaccinated with mRNA-1273, had 259 increased reactivity to PEG, particularly after the boost (Fig. 2E) , but there was no increase in 260 antibody titers following BNT162b2 administration (Fig. 2F) . Overall, these results suggest that 261 the antibodies induced towards the vaccine formulation components may be directed against 262 PEG, which is present in the vaccine formulation. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101/2022.04.15.22273914 doi: medRxiv preprint performed. We found that the mRNA-1273 induced antibodies detected against the BNT162b2 268 ( Fig. 3A ) or mRNA-1273 ( Fig. 3B ) vaccine formulations not only correlated well with PEG as 269 measured by using PEGylated BSA, but a correlation was observed between the PEGylated-BSA 270 AUC values and the irrelevant mRNA-LNPs (Fig. 3C) , as well as with the low molecular weight PEG 271

(3.35kDa) AUCs (Fig. 3D) . Moreover, the independent correlation of PEGylated-BSA AUC values 272 vs the BNT162b2 or mRNA-1273 formulation, the irrelevant mRNA-LNPs, or the low molecular 273 weight PEG AUCs, increased in a prime-boost dependent manner, with the lowest correlation 274 observed at baseline, and increasing correlations after the prime, followed by high correlations 275 after the boost ( Supplementary Fig. 1) . The absolute values of the geometric mean AUCs for every 276 ELISA and the fold induction after the prime or the boost are shown in Table 2 To explore if vaccinees displayed other classes of anti-PEG antibodies, we measured the reactivity 282 of IgM using the PEGylated BSA based ELISA. Similar to the IgG pattern previously observed, we 283 detected PEG-specific IgM -although at low levels -in the mRNA-1273 recipients in a vaccination 284 dependent manner (Fig. 4B) , however no induction of IgM in the BNT162b2 vaccinees (Fig. 4A) . 285

Moreover, we assessed whether individuals could potentially induce IgE antibodies directed to 286 PEG in response to vaccination, however levels of PEG-specific IgE were undetectable in all the 287 participants, irrespective of the vaccine type received (Figs. 4C and 4D). As a control for IgE 288 detection, we used plates coated with house dust mite (HDM) antigens, which allowed detecting 289 IgE in some of the participants (Supplementary Fig. 2) . Moreover, as a control to ensure that the 290 anti-PEG antibodies detected through this work were directed specifically against PEG, and 291 exclude the possibility that the BSA contained in the PEG-BSA reagent was as a target of the 292 reactivity detected, we performed ELISAs in plates pre-coated with 1% BSA (Figs. 4E and 4F) . We 293 detected residual reactivity in three individuals, although at levels very close to the limit of 294 detection. These residual antibodies were, however, detected in both the BNT162b2 and mRNA-295 1273 groups, and were irrespective of the vaccination time point. Overall, these results indicate 296 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. reactions or other types of adverse reactions following vaccination, mounted differential anti-304 PEG antibodies at baseline or after vaccination, we used samples from a selection of study 305 participants that reported vaccine-associated side effects such as delayed onset reactions 306 including injection site rashes (N=8) or severe allergic reaction (N=1). Overall, although levels of 307 anti-PEG antibodies were slightly higher at baseline, we did not find a significant association 308 between baseline anti-PEG titers and antibody induction after vaccination with mRNA-1273 or 309

BNT162b2. However, individuals receiving the mRNA-1273 formulation (Fig. 5B) , but not the ones 310 receiving the BNT162b2 vaccine (Fig. 5A) , induced significantly higher levels of anti-PEG 311 antibodies in a vaccination-dependent manner, similar to the findings described above. In 312 summary, these findings indicate that although there is an increase in the anti-PEG antibodies in 313 the mRNA-1273 vaccinees, there was no obvious association between PEG antibodies and 314 adverse reactions. Pre-existing anti-PEG levels are not associated with a more robust PEG 315 antibody induction following vaccination .  316   317  318  319  320  321  322  323  324  325  326  327  328  329  330 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The pre-existing antibody levels against PEG could be due to previous exposures to PEGylated 357 drugs (14, [25] [26] [27] or PEG-containing products (23). Importantly, we assessed the presence of 358 spike-or PEG-specific IgE antibodies and we did not find detectable levels of IgE antibodies, 359 including in the one participant who experienced a severe allergic reaction in response to 360 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101/2022.04.15.22273914 doi: medRxiv preprint 13 vaccination. Our results are in line with previous findings detecting PEG-specific IgG following 361 vaccination, but a lack of IgE (29). Allergy skin testing to PEG also was negative (unpublished 362 data). Immediate allergic reactions following vaccination, such anaphylaxis, are likely to be 363 mediated by IgE-independent mechanisms of diverse nature (29), and the relevance of PEG-364 specific IgG induced by vaccination remains to be investigated. Interestingly, via a genome-wide 365 association study, an immunoglobulin heavy chain (IGH) locus has been associated with the anti-366 PEG IgM response (30). Although such association was not present for IgG, IGH polymorphisms 367 associated with switched anti-PEG IgG subsets require further exploration. Hence, it is unlikely that the differential patterns of anti-PEG antibodies detected in mRNA-1273 378 vs BNT162b2 vaccinee's sera, are due to PEG structural differences in the formulations, but 379 rather, this might be the result of the higher dose of mRNA given to mRNA-1273 vaccine 380 recipients -100μg for mRNA-1273 vs 30μg for BNT162b2, the result of the higher PEGylated lipid 381 dose in mRNA-1273 or the way PEG is presented by the carrier lipid (35). Moreover, serum from 382 mRNA-1273 vaccine recipients was able to recognize components of both formulations in a 383 prime-boost dependent manner. It remains to be explored whether the anti-PEG antibodies 384 induced following vaccination are directed towards the backbone of the PEG molecule, or the 385 methoxy group present in PEG from both formulations. Overall, our study reports the induction 386 of PEG antibodies following administration of one of the currently used mRNA-based vaccine 387

formulation. The clinical relevance of PEG-reactive antibodies induced by mRNA-1273 388 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. ; 544  545  546  547  548  549  550  551  552  553  554  555  556  557  558  559  560  561  562  563  564 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 570  571  572  573  574  575  576  577  578  579  580  581  582  583  584  585  586  587  588 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 for BNT162b2 and n=10 for mRNA-1273 groups), 18.9 days (arithmetic mean ±2.4 SD) after the 623 prime (n=10 for BNT162b2 and n=10 for mRNA-1273 groups) and 19.3 days (arithmetic mean 624 ±3.9 SD) after the boost (n=10 for BNT162b2 and n=10 for mRNA-1273 groups), were tested for 625

IgM (A and B) or IgE (C and D) antibodies against PEGylated BSA 20kDa or against bovine serum 626 albumin (BSA, E and F). Statistically significant differences between post-prime/post-boost vs 627 baseline antibody levels are shown. One-way ANOVA with Tukey's multiple comparisons test. *, 628 P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 

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Induced and pre-463 existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for 464 refractory gout, including in organ transplant recipients

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Evidence for

. CC-BY-NC-ND 4.0 International license It 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) 21 reaction. Sera from BNT162b2 (A) or mRNA-1273 (B) vaccinees was collected at baseline (n=28 633for A and C, and n=19 for B and D), 14.5 days (arithmetic mean ±4.8 SD) after the prime (n=23 634for A and C, and n=17 for B and D) or 25.2 days (arithmetic mean ±11.6 SD) after the boost (n=26 635for A and C and n=17 for B and D), and tested for IgG (A and B) , IgM (C and D) or IgE (E and F) 636antibodies against 20kDa PEGylated BSA by ELISA. Dotted line represents the limit of detection 637(LoD) of the assay. Statistically significant differences between post-prime/post-boost vs baseline 638 antibody levels are shown. One-way ANOVA with Tukey's multiple comparisons test. *, P < 0.05; 639 **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The positive control used for measurement of antigen-specific IgE is shown in A. The positive 650control used for measurement of BSA specific IgG is shown in B. 651 . CC-BY-NC-ND 4.0 International license It 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)