The BNT162b2 mRNA vaccine against SARS-CoV-2 reprograms both adaptive and innate immune responses

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.


Main text
Coronavirus disease 2019  is a new respiratory tract infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread worldwide since the end of 2019 causing a global pandemic.The COVID-19 pandemic represents the most important healthcare crisis humanity has encountered since World War II, combined with a devastating societal and economic impact.Confronted with this critical situation, a major effort to develop vaccines against COVID-19 has been initiated in many countries around the world.
To date, 13 vaccines have been approved for use in humans ("COVID-19 vaccine tracker | RAPS," n.d.).The scale of the pandemic has led to accelerated development of vaccines based on new technologies, such as mRNA-and viral vector-based vaccines (van Riel & de Wit, 2020).
One of the most widely used anti-COVID-19 vaccines in the world was developed by a collaboration between BioNTech and Pfizer (BNT162b2).This vaccine is based on a lipid nanoparticle-formulated, nucleoside-modified mRNA that encodes a prefusion stabilized form of the spike (S)-protein derived from the SARS-CoV-2 strain isolated early on in Wuhan, China (Walsh et al., 2020).Several phase-3 trials have demonstrated that BNT162b2 elicits broad humoral and cellular responses, providing protection against COVID-19 (Sahin et al., 2020;Walsh et al., 2020).
While global vaccination campaigns against the SARS-CoV-2 infection are rolled out, major challenges remain, especially the spread of novel virus variants (Madhi et al., 2021).One of the most prominent mutations during the pandemic has been the spike D614G substitution to the Wuhan Hu-1 original strain (Korber et al., 2020).With the steadily increasing prevalence of .infections, SARS-CoV-2 variants emerged with multiple spike mutations and were first detected in the United Kingdom (B.1.1.7 lineage), South Africa (B.1.351lineage), and Brazil (P.1 lineage).
These variants are of significant concern because of their potential effects on disease severity, viral transmissibility, reinfection rates, and vaccine effectiveness (Abdool Karim & de Oliveira, 2021).
The capacity of BNT162b2 to induce effective humoral and cellular immunity against the new SARS-CoV-2 variants is only now beginning to be understood.Whereas neutralization of B.1.1.7 was either similar or just slightly reduced as compared to the standard strain (Muik et al., 2021;Wang, Nair, et al., 2021), neutralizing titers of B.1.351were markedly diminished (Liu et al., 2021;Planas et al., 2021;Wang, Nair, et al., 2021) after vaccination of healthy volunteers with BNT162b2.In contrast, cellular immunity against the virus variants seems to be less affected (Lilleri et al., 2021;Skelly et al., 2021).In addition, an unexplored area is whether vaccination with BNT162b2 also leads to long-term effects on innate immune responses: this could be very relevant in COVID-19, in which dysregulated inflammation plays an important role in the pathogenesis and severity of the disease (Tahaghoghi-Hajghorbani et al., 2020).The long-term modulation of innate immune responses has been an area of increased interest in the last years: multiple studies have shown that long-term innate immune responses can be either increased (trained immunity) or down-regulated (innate immune tolerance) after certain vaccines or infections (Netea et al., 2020).
In this study, we assessed the effect of the BNT162b2 mRNA COVID-19 vaccine on both the innate and adaptive (humoral and cellular) immune responses.We first examined the concentration of RBD-and S-binding antibody isotype concentrations before vaccination .
(baseline; t1), 3 weeks after the first dose of 30 μg of BNT162b2 (t2), and two weeks after the second dose (t3) (Figure S1A).We calculated fold-changes by comparing concentrations from both t2 and t3 to baseline.BNT162b2 vaccination elicited high anti-S protein and anti-RBD antibody concentrations already after the first vaccination, and even stronger responses after the second dose of the vaccine.As expected, IgG responses were the most pronounced, with RBD-specific median fold changes at t2 and t3 of 56-fold and 1839-fold, and S-specific fold changes of 208-fold and 1100-fold, respectively.The lowest observed fold change increase to pre-vaccination levels of IgG targeting RBD was 14-fold at t2, and 21-fold at t3, respectively.For S-specific IgG, the fold changes were at least 32-fold at t2 and 339-fold at t3. Regarding IgA concentrations, a single dose of the vaccine elicited a 7-fold increase in the RBD-specific concentration and a 35-fold increase in the S-specific concentration.The second dose enhanced the antibody concentrations elicited by the first vaccination by 24-fold and 52-fold, for RBD and S, respectively.Compared to IgG and IgA, the increase in IgM concentrations was considerably lower.RBD-specific concentration only doubled after the first dose, and it did not further increase after the second dose of the vaccine.In contrast, S-specific fold changes were 11-fold at t2 and 20-fold at t3 (Figure S1A).These results confirm and extend recent observations reporting strong induction of humoral responses by BNT162b2 vaccination (Sahin et al., 2020).
Similar to our investigation, several studies reported 6 to 14-fold decreased neutralizing activity of post-vaccine sera against the B.1.351variant, and only slightly reduced activity against B.1.1.7,when compared to the standard strain (Planas et al., 2021;Shen et al., 2021;Wang, Liu, et al., 2021).These data support the evidence that B.1.351,and possibly other variants, may be able to escape vaccine-induced humoral immunity to a certain extent (Kustin et al., 2021).Furthermore, the PRNT titer and the antibody concentrations of IgG after the second dose were strongly correlated (Figure S1C).The correlation was stronger for B. 1.1.7 and B.1.351than for the standard strain, both for anti-RBD and anti-S.
BNT162b2 vaccination has been reported to activate virus-specific CD4+ and CD8+ T cells, and upregulate the production of immune-modulatory cytokines such as IFN-γ (Sahin et al., 2020).(Tarke et al., 2021).IFN-γ production was higher by at least 50% in 37.5% of the subjects upon stimulation with the standard SARS-CoV-2 strain, in 50% of the subjects upon stimulation with the B.1.1.7 variant and the B.1.351variant, but only in 18,75% of the subjects upon stimulation with the Bavarian variant (Figure S1D).These findings argue that BNT162b2 vaccination induces better humoral than cellular immune responses.Weak Tcell responses have previously been reported in vaccinees that have received just a single dose . of BNT162b2 (Prendecki et al., 2021;Stankov, Cossmann, Bonifacius, Dopfer-jablonka, & Morillas, 2021).Intriguingly, the best cellular responses after vaccination were against the B.1.351variant: the fact that the neutralizing antibody responses against this variant were relatively poor, that may raise the possibility that the protective BNT162b2 vaccine effects against this variant may be mainly reliant on cellular, rather than humoral, responses.No significant differences between the individual variants were observed.The absolute concentrations of the cytokines after stimulations can be found in Supplementary Table 2. Interestingly, we observed important heterologous effects of BNT162b2 vaccination on IFN-γ production induced by other stimuli as well (Figures S2E, 2F).BNT162b2 vaccination decreased IFN-γ production upon stimulation with the TLR7/8 agonist R848 (Figure S2F).In contrast, the IFN-γ production induced by inactivated influenza virus tended to be higher two weeks after the second BNT162b2 vaccination, though the differences did not reach statistical significance.
We did not find any significant correlation between cellular responses and IgG antibody titers.
Besides their effects on specific (adaptive) immune memory, certain vaccines such as Bacillus Calmette-Guérin (BCG) and the measles, mumps, and rubella (MMR) vaccine also induce longterm functional reprogramming of cells of the innate immune system.(Netea et al., 2020).This biological process is also termed trained immunity when it involves increased responsiveness, or innate immune tolerance when it is characterized by decreased cytokine production (Ifrim et al., 2014).Although these effects have been proven mainly for live attenuated vaccines, we sought to investigate whether the BNT162b2 vaccine might also induce effects on innate immune responses against different viral, bacterial and fungal stimuli.One of the trademarks of trained immunity is an elevated production of inflammatory cytokines following a secondary .insult (Quintin et al., 2012).Surprisingly, the production of the monocyte-derived cytokines TNF-α, IL-1β and IL-1Ra tended to be lower after stimulation of PBMCs from vaccinated individuals with either the standard SARS-CoV-2 strain or heterologous Toll-like receptor ligands (Figures 1 and 2).TNF-α production (Figure 1B-1G) following stimulation with the TLR7/8 agonist R848 of peripheral blood mononuclear cells from volunteers was significantly decreased after the second vaccination (Figure 1C).The same trend was observed after stimulation with the TLR3 agonist poly I:C (Figure 1D), although the difference did not reach statistical significance.In contrast, the responses to the fungal pathogen Candida albicans were higher after the first dose of the vaccine (Figure 1G).The impact of the vaccination on IL-1β production was more limited (Figure 2A-2F), though the response to C. albicans was significantly increased (Figure 2F).The production of the anti-inflammatory cytokine IL-1Ra (Figure 2G The induction of tolerance towards stimulation with TLR7/8 (R848) or TLR4 (LPS) ligands by BNT162b2 vaccination may indicate a more balanced inflammatory reaction during infection with SARS-CoV-2, and one could speculate whether such effect may be thus useful to regulate the potential over-inflammation in COVID-19, one of the main causes of death (Tang et al., 2020).On the other hand, inhibition of innate immune responses may diminish anti-viral responses.Type I interferons also play a central role in the pathogenesis and response against viral infections, including COVID-19 (Hadjadj et al., 2020).With this in mind, we also assessed the production of IFN-α by immune cells of the volunteers after vaccination.Although the .concentrations of IFN-α were below the detection limit of the assay for most of the stimuli, we observed a significant reduction in the production if IFN-α secreted after stimulation with poly I:C and R848 after the administration of the second dose of the vaccine (Figure 1H, 1I).This may hamper the initial innate immune response against the virus, as defects in TLR7 have been shown to result in and increased susceptibility to COVID-19 in young males (Van Der Made et al., 2020).These results collectively demonstrate that the effects of the BNT162b2 vaccine go beyond the adaptive immune system and can also modulate innate immune responses.
The effect of the BNT162b2 vaccination on innate immune responses may also indicate a potential to interfere with the responses to other vaccinations, as known for other vaccines to be as 'vaccine interference' (Lum et al., 2010;Nolan et al., 2008;Vajo, Tamas, Sinka, & Jankovics, 2010).Future studies are therefore needed to investigate this possibility, especially the potential interaction with the influenza vaccine: in the coming years (including the autumn of 2021) COVID-19 vaccination programs will probably overlap with the seasonal Influenza vaccination, so it is crucial to perform additional studies to elucidate the potential interactions and effects of the COVID-19 vaccines with the current vaccination schedules, especially for immunosuppressed and elderly individuals.
The generalizability of these results is subject to certain limitations.First, the number of volunteers in this study was relatively small, although in line with earlier immunological studies on the effects of COVID-19 vaccines.Second, our cohort consisted of healthcare workers, who are middle-aged and healthy, and future studies in elderly individuals and people with comorbidities and other underlying risk factors for severe COVID-19 infections need to be performed (Gao et al., 2021).Third, our study is performed only with individuals with a Western .European ancestry.Therefore, the conclusions of our study should be tested in populations with different ancestry and alternative lifestyles since the induction of innate and adaptive immune responses is largely dependent on different factors such as genetic background, diet, and exposure to environmental stimuli which largely differ between communities around the globe.
In conclusion, our data show that the BNT162b2 vaccine induces effects on both the adaptive and the innate branch of immunity and that these effects are different for various SARS-CoV-2 strains.Intriguingly, the BNT162b2 vaccine induces reprogramming of innate immune responses as well, and this needs to be taken into account: in combination with strong adaptive immune responses, this could contribute to a more balanced inflammatory reaction during COVID-19 infection, or it may contribute to a diminished innate immune response towards the virus.BNT162b2 vaccine is clearly protective against COVID-19, but the duration of this protection is not yet known, and one could envisage future generations of the vaccine incorporating this knowledge to improve the range and duration of the protection.Our findings need to be confirmed by conducting larger cohort-studies with populations with diverse backgrounds, while further studies should examine the potential interactions between BNT162b2 and other vaccines.   .cat #51985034).Afterwards the concentrated virus on the filter was diluted back to the original volume using Opti-MEM and the purified viral aliquots were stored at -80 °C.The infectious viral titers were measured using plaque assays as described (Varghese et al., 2021) and stocks were heat inactivated for 60 min at 56 °C for use in stimulation experiments.

Measurement of antibody levels against RBD and Spike protein
To measure the levels of antibodies against RBD and Spike protein, a fluorescent-bead-based multiplex immunoassay (MIA) was developed as previously described by (Fröberg et al., 2021).
The stabilized pre-fusion conformation of the ectodomain of the Spike protein (amino acids 1 -1,213) fused with the trimerization motif GCN4 (S-protein) and the receptor binding domain of the S-protein (RBD) were each coupled to beads or microspheres with distinct fluorescence excitation and emission spectra.Serum samples were diluted and incubated with the antigencoupled microspheres.Following incubation, the microspheres were washed and incubated with phycoerythrin-conjugated goat anti-human, IgG, IgA and IgM.The data were acquired on the Luminex FlexMap3D System.Mean Fluorescent Intensities (MFI) were converted to arbitrary units (AU/ml) by interpolation from a log-5PL-parameter logistic standard curve and log-log axis transformation, using Bioplex Manager 6.2 (Bio-Rad Laboratories) software.

Statistical analysis
Graphpad Prism 8 was used for all statistical analyses.Outcomes between paired groups were analyzed by Wilcoxon's matched-pairs signed-rank test.Three or more groups were compared using Kruskal-Wallis Test -Dunnet's multiple comparison.P-value of less than 0.05 .
Hence, we assessed IFN-γ secretion from peripheral blood mononuclear cells (PBMCs) before and after BNT162b2 vaccination in response to heat-inactivated SARS-CoV-2 strains (Figs 2A-2D).While vaccination with BNT162b2 generally seems to moderately increase specific IFN-γ production after the second dose of the vaccine, this reached statistical significance only upon stimulation with B.1.351variant (Figure S2A-2C).The same tendency has also been observed by Tarke et al. who used synthetic SARS-CoV-2 variants proteins to induce elevated IFN-γ responses against B.1.351 -2L) was reduced in response to bacterial lipopolysaccharide (LPS) and C. albicans after the second vaccination (Figure 2K, 2L), which is another argument for a shift towards stronger inflammatory responses to fungal stimuli after vaccination.IL-6 responses were similarly decreased, though less pronounced (data not shown).

Figure 1 .
Figure 1.TNF-α and IFN-α production in response to heterologous stimuli in PBMCs isolated from vaccinated subjects.(A) Description of the study: vaccination and blood collection days.(B-G) Fold change values of TNF-α production are calculated individually for each subject by division of t2:t1 and t3:t1.Data are presented as fold changes ± SEM (n=15-16) and analysed by Wilcoxon's matched-pairs signed-rank test comparing each ratio to t1=1 (red line).(H-I) IFN-α production (pg/ml) at t1, t2 and t3.Data are presented as cytokine concentration ± SEM (n=15-16) and analysed by Wilcoxon's matched-pairs signed-rank test.

Figure 2 .
Figure 2. IL-1β and IL-1Ra production in response to heterologous stimuli in PBMCs isolated from vaccinated subjects.IL-1β (A-F) and IL-1Ra (G-L) Fold change values are calculated individually for each subject by division of t2:t1 and t3:t1.Data are presented as fold changes ± SEM (n=15-16) and analysed by Wilcoxon's matched-pairs signed-rank test comparing each ratio to t1=1 (red line).