key: cord-0793846-djev6er7
authors: Fohse, F. K.; Geckin, B.; Overheul, G. J.; van de Maat, J.; Kilic, G.; Bulut, O.; Dijkstra, H.; Lemmers, H.; Sarlea, S. A.; Reijnders, M.; Hoogerwerf, J.; ten Oever, J.; Simonetti, E.; van de Veerdonk, F. L.; Joosten, L. A. B.; Haagmans, B. L.; van Crevel, R.; Li, Y.; van Rij, R. P.; GeurtsvanKessel, C.; de Jonge, M. I.; Dominguez-Andres, J.; Netea, M. G.
title: The BNT162b2 mRNA vaccine against SARS-CoV-2 reprograms both adaptive and innate immune responses
date: 2021-05-06
journal: nan
DOI: 10.1101/2021.05.03.21256520
sha: 4592dd21b760da4fe3d402ca8d92f9c57cffc8e2
doc_id: 793846
cord_uid: djev6er7

The mRNA-based BNT162b2 vaccine from Pfizer/BioNTech was the first registered COVID-19 vaccine and has been shown to be up to 95% effective in preventing SARS-CoV-2 infections. Little is known about the broad effects of the new class of mRNA vaccines, especially whether they have combined effects on innate and adaptive immune responses. Here we confirmed that BNT162b2 vaccination of healthy individuals induced effective humoral and cellular immunity against several SARS-CoV-2 variants. Interestingly, however, the BNT162b2 vaccine also modulated the production of inflammatory cytokines by innate immune cells upon stimulation with both specific (SARS-CoV-2) and non-specific (viral, fungal and bacterial) stimuli. The response of innate immune cells to TLR4 and TLR7/8 ligands was lower after BNT162b2 vaccination, while fungi-induced cytokine responses were stronger. In conclusion, the mRNA BNT162b2 vaccine induces complex functional reprogramming of innate immune responses, which should be considered in the development and use of this new class of vaccines.

on new technologies, such as mRNA-and viral vector-based vaccines (van Riel & de Wit, 2020) . 67

One of the most widely used anti-COVID-19 vaccines in the world was developed by a 68 collaboration between BioNTech and Pfizer (BNT162b2). This vaccine is based on a lipid 69 nanoparticle-formulated, nucleoside-modified mRNA that encodes a prefusion stabilized form 70 of the spike (S)-protein derived from the SARS-CoV-2 strain isolated early on in Wuhan, China 71 (Walsh et al., 2020) . Several phase-3 trials have demonstrated that BNT162b2 elicits broad 72 humoral and cellular responses, providing protection against COVID-19 (Sahin et al., 2020; 73 Walsh et al., 2020) . 74 75 While global vaccination campaigns against the SARS-CoV-2 infection are rolled out, major 76 challenges remain, especially the spread of novel virus variants (Madhi et al., 2021) . One of the 77 most prominent mutations during the pandemic has been the spike D614G substitution to the 78 Wuhan Hu-1 original strain (Korber et al., 2020) . With the steadily increasing prevalence of 79 . 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 May 6, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 infections, SARS-CoV-2 variants emerged with multiple spike mutations and were first detected 80 in the United Kingdom (B.1.1.7 lineage), South Africa (B.1.351 lineage), and Brazil (P.1 lineage). 81

These variants are of significant concern because of their potential effects on disease severity, 82 viral transmissibility, reinfection rates, and vaccine effectiveness (Abdool Karim & de Oliveira, 83 2021) . 84

The capacity of BNT162b2 to induce effective humoral and cellular immunity against the new 86 SARS-CoV-2 variants is only now beginning to be understood. Whereas neutralization of B.1.1.7 87 was either similar or just slightly reduced as compared to the standard strain (Muik et al., 2021; 88 Wang, Nair, et al., 2021) , neutralizing titers of B.1.351 were markedly diminished (Liu et al., 89 2021; Planas et al., 2021; Wang, Nair, et al., 2021) 

BNT162b2. In contrast, cellular immunity against the virus variants seems to be less affected 91 (Lilleri et al., 2021; Skelly et al., 2021) . In addition, an unexplored area is whether vaccination 92 with BNT162b2 also leads to long-term effects on innate immune responses: this could be very 93 relevant in COVID-19, in which dysregulated inflammation plays an important role in the 94 pathogenesis and severity of the disease (Tahaghoghi-Hajghorbani et al., 2020) . The long-term 95 modulation of innate immune responses has been an area of increased interest in the last 96 years: multiple studies have shown that long-term innate immune responses can be either 97 increased (trained immunity) or down-regulated (innate immune tolerance) after certain 98 vaccines or infections (Netea et al., 2020) . 99

In this study, we assessed the effect of the BNT162b2 mRNA COVID-19 vaccine on both the 101 innate and adaptive (humoral and cellular) immune responses. We first examined the 102 concentration of RBD-and S-binding antibody isotype concentrations before vaccination 103 . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint 5 (baseline; t1), 3 weeks after the first dose of 30 μg of BNT162b2 (t2), and two weeks after the 104 second dose (t3) ( Figure S1A ). We calculated fold-changes by comparing concentrations from 105 both t2 and t3 to baseline. BNT162b2 vaccination elicited high anti-S protein and anti-RBD 106 antibody concentrations already after the first vaccination, and even stronger responses after 107 the second dose of the vaccine. As expected, IgG responses were the most pronounced, with 108 RBD-specific median fold changes at t2 and t3 of 56-fold and 1839-fold, and S-specific fold 109 changes of 208-fold and 1100-fold, respectively. The lowest observed fold change increase to 110 pre-vaccination levels of IgG targeting RBD was 14-fold at t2, and 21-fold at t3, respectively. For 111 S-specific IgG, the fold changes were at least 32-fold at t2 and 339-fold at t3. Regarding IgA 112 concentrations, a single dose of the vaccine elicited a 7-fold increase in the RBD-specific 113 concentration and a 35-fold increase in the S-specific concentration. The second dose 114 enhanced the antibody concentrations elicited by the first vaccination by 24-fold and 52-fold, 115

for RBD and S, respectively. Compared to IgG and IgA, the increase in IgM concentrations was 116 considerably lower. RBD-specific concentration only doubled after the first dose, and it did not 117 further increase after the second dose of the vaccine. In contrast, S-specific fold changes were 118 11-fold at t2 and 20-fold at t3 ( Figure S1A ). These results confirm and extend recent 119 observations reporting strong induction of humoral responses by BNT162b2 vaccination (Sahin 120 et al., 2020) . 121

To investigate the neutralizing capacity of the serum against SARS-CoV-2 variants, we 123 performed 50% plaque reduction neutralization testing (PRNT50) using sera collected two 124 weeks after the second vaccine administration. All the serum samples neutralized the D614G 125 strain and the B.1.1.7 variant with titers of at least 1:80. However, six subjects (37,5%) had titers 126 lower than 1:80 against the B.1.351 variant. Geometric mean neutralizing titers against the 127 . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint 6 D614G strain, B.1.1.7 and B.1.351 were 381, 397, and 70, respectively (Figure S1B, p<0.001). 128 Similar to our investigation, several studies reported 6 to 14-fold decreased neutralizing activity 129 of post-vaccine sera against the B.1.351 variant, and only slightly reduced activity against 130 B.1.1.7, when compared to the standard strain (Planas et al., 2021; Shen et al., 2021; Wang, 131 Liu, et al., 2021) . These data support the evidence that B.1.351, and possibly other variants, 132 may be able to escape vaccine-induced humoral immunity to a certain extent (Kustin et al., 133 2021) . Furthermore, the PRNT titer and the antibody concentrations of IgG after the second 134 dose were strongly correlated ( Figure S1C ). The correlation was stronger for B.1.1.7 and B.1.351 135 than for the standard strain, both for anti-RBD and anti-S. 136 137 BNT162b2 vaccination has been reported to activate virus-specific CD4+ and CD8+ T cells, and 138 upregulate the production of immune-modulatory cytokines such as IFN-γ (Sahin et al., 2020) . 139

Hence, we assessed IFN-γ secretion from peripheral blood mononuclear cells (PBMCs) before 140 and after BNT162b2 vaccination in response to heat-inactivated SARS-CoV-2 strains ( 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 May 6, 2021. IFN-γ production upon stimulation with the TLR7/8 agonist R848 ( Figure S2F ). In contrast, the 162 IFN-γ production induced by inactivated influenza virus tended to be higher two weeks after 163 the second BNT162b2 vaccination, though the differences did not reach statistical significance. 164

We did not find any significant correlation between cellular responses and IgG antibody titers. 165

Besides their effects on specific (adaptive) immune memory, certain vaccines such as Bacillus 167

Calmette-Guérin (BCG) and the measles, mumps, and rubella (MMR) vaccine also induce long-168 term functional reprogramming of cells of the innate immune system. (Netea et al., 2020) . This 169 biological process is also termed trained immunity when it involves increased responsiveness, 170 or innate immune tolerance when it is characterized by decreased cytokine production (Ifrim 171 et al., 2014) . Although these effects have been proven mainly for live attenuated vaccines, we 172 sought to investigate whether the BNT162b2 vaccine might also induce effects on innate 173

immune responses against different viral, bacterial and fungal stimuli. One of the trademarks 174 of trained immunity is an elevated production of inflammatory cytokines following a secondary 175 . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint 8 insult (Quintin et al., 2012) . Surprisingly, the production of the monocyte-derived cytokines 176 TNF-α, IL-1β and IL-1Ra tended to be lower after stimulation of PBMCs from vaccinated 177 individuals with either the standard SARS-CoV-2 strain or heterologous Toll-like receptor 178 ligands (Figures 1 and 2) . TNF-α production ( Figure 1B-1G) following stimulation with the 179 TLR7/8 agonist R848 of peripheral blood mononuclear cells from volunteers was significantly 180 decreased after the second vaccination ( Figure 1C ). The same trend was observed after 181 stimulation with the TLR3 agonist poly I:C ( Figure 1D ), although the difference did not reach 182 statistical significance. In contrast, the responses to the fungal pathogen Candida albicans were 183 higher after the first dose of the vaccine ( Figure 1G ). The impact of the vaccination on IL-1β 184 production was more limited ( We thank all the volunteers to the study for their willingness to participate. Purified Spike 245 protein and the receptor binding domain of the Spike protein (RBD) were kindly provided by 246 . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint IFN-α production (pg/ml) at t1, t2 and t3. Data are presented as cytokine concentration ± SEM 270 (n=15-16) and analysed by Wilcoxon's matched-pairs signed-rank test. 271 . 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 May 6, 2021. Table 1) . Key exclusion criteria included a medical history of COVID-19. Sera and 299 blood samples were collected before the first administration of BNT162b2, three weeks after 300 the first dose (right before the second dose), and two weeks after the second dose. A high 301 . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint percentage (56.3%) of individuals had been vaccinated with BCG in the past 12 months due to 302 the fact that many participants participated in parallel in a BCG-trial. One individual was 303 removed from the dataset after detecting high concentrations of antibodies against SARS-CoV-304 2 N-antigen at baseline. Ultra-15 column with 100 kDa cutoff (Sigma-Aldrich, Germany, cat #UFC910008), which was 324 washed 3 times using Opti-MEM supplemented with GlutaMAX (Thermo Fisher Scientific, USA, 325 . 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 May 6, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 cat #51985034). Afterwards the concentrated virus on the filter was diluted back to the original 326 volume using Opti-MEM and the purified viral aliquots were stored at -80 °C. The infectious 327 viral titers were measured using plaque assays as described (Varghese et al., 2021) and stocks 328 were heat inactivated for 60 min at 56 °C for use in stimulation experiments. 329 330

To measure the levels of antibodies against RBD and Spike protein, a fluorescent-bead-based 332 multiplex immunoassay (MIA) was developed as previously described by (Fröberg et al., 2021) . . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint were 2-fold diluted in Dulbecco modified Eagle medium supplemented with NaHCO3, HEPES 350 buffer, penicillin, streptomycin, and 1% fetal bovine serum, starting at a dilution of 1:10 in 60 351 μL. We then added 60 μL of virus suspension (400 plaque-forming units) to each well and 352 incubated at 37°C for 1h. After 1 hour incubation, we transferred the mixtures on to Vero-E6 353 cells and incubated for 8 hours. After incubation, we fixed the cells with 10% formaldehyde 354 and stained the cells with polyclonal rabbit anti-SARS-CoV antibody (Sino Biological) and a 355 secondary peroxidase-labeled goat anti-rabbit IgG (Dako). We developed signal by using a 356 precipitate forming 3,3′,5,5′-tetramethylbenzidine substrate (True Blue; Kirkegaard and Perry 357

Laboratories) and counted the number of infected cells per well by using an ImmunoSpot 358

Image Analyzer (CTL Europe GmbH). The serum neutralization titer is the reciprocal of the 359 highest dilution resulting in an infection reduction of >50% (PRNT50). We considered a titer 360 >20 to be positive based on assay validation 361 362

Blood samples from subjects were collected into EDTA-coated tubes (BD Bioscience, USA) and 364 used as the source of peripheral blood mononuclear cells (PBMCs) after sampling sera from 365 each individual. Blood is diluted 1:1 with PBS (1X) without Ca++, Mg++ (Westburg, The 366

Netherlands, cat #LO BE17-516F) and PBMCs were isolated via density gradient centrifuge 367 using Ficoll-PaqueTM-plus (VWR, The Netherlands, cat #17-1440-03P). The tubes used for the 368 isolation was specialized SepMate-50 tubes (Stem Cell Technologies, cat #85450) to ensure 369 better separation. Cells counts were determined via Sysmex XN-450 hematology analyzer. 370

Afterwards, PBMCs were frozen using Recovery Cell Culture Freezing (Thermo Fisher 371

Scientific, USA, cat #12648010) in the concentration of 15x106/mL. 372 373 . 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 May 6, 2021. heat-inactivated SARS-CoV-2 Wuhan Hu-1 strain (3.3x10 3 TCID50/mL), SARS-CoV-2 B.1.1.7 384 (3.3x10 3 TCID50/mL), SARS-CoV-2 B.1.351 (3.3x10 3 TCID50/mL), and SARS-CoV-2 Bavarian 385 (3.3x10 3 TCID50/mL) variants, Influenza (3.3x10 5 TCID50/mL), 10 µg/mL Poly I:C (Invivogen, 386 USA, cat #tlrl-pic), 3 µg/mL R848 (Invivogen, USA, cat #tlrl-r848), 10 ng/mL E. coli LPS, and 1 x 387 10 6 /mL C. albicans. The PBMCs were incubated with the stimulants for 24 hours to detect IL-388 1β, TNF-α, IL-6, IL-1Ra and 7 days to detect IFN-γ. Supernatants were collected and stored in -389 20°C. Secreted cytokine levels from supernatants were quantified by ELISA (IL-1β cat # DLB50, 390 TNF-α cat # STA00D, IL-6 cat # D6050, IL-1Ra cat # DRA00B, IFN-γ cat #DY285B, R&D Systems, 391 USA). 392 393

Graphpad Prism 8 was used for all statistical analyses. Outcomes between paired groups were 395 analyzed by Wilcoxon's matched-pairs signed-rank test. Three or more groups were 396 compared using Kruskal-Wallis Test -Dunnet's multiple comparison. P-value of less than 0.05 397 . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint was considered statistically significant. Spearman correlation was used to determine 398 correlation between groups. 399 400 401 402 403 404 . 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 May 6, 2021. ; https://doi.org/10.1101/2021.05.03.21256520 doi: medRxiv preprint

New SARS-CoV-2 Variants -Clinical

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