key: cord-0835374-7z6ft3jh authors: Sahin, U.; Muik, A.; Derhovanessian, E.; Vogler, I.; Kranz, L. M.; Vormehr, M.; Baum, A.; Pascal, K.; Quandt, J.; Maurus, D.; Brachtendorf, S.; Loerks, V. L.; Sikorski, J.; Hilker, R.; Becker, D.; Eller, A.-K.; Gruetzner, J.; Boesler, C.; Rosenbaum, C.; Kuehnle, M.-C.; Luxemburger, U.; Kemmer-Brueck, A.; Langer, D.; Bexon, M.; Bolte, S.; Kariko, K.; Palanche, T.; Fischer, B.; Schultz, A.; Shi, P.-Y.; Fontes-Garfias, C.; Perez, J. L.; Swanson, K. A.; Loschko, J.; Scully, I. L.; Cutler, M.; Kalina, W.; Kyratsous, C. A.; Cooper, D.; Dormitzer, P. R.; Jansen, K. U.; Tuereci, O. title: Concurrent human antibody andTH1 type T-cell responses elicited by a COVID-19 RNA vaccine date: 2020-07-20 journal: nan DOI: 10.1101/2020.07.17.20140533 sha: cd0aa536da6b2bb59ecae8b10889fbc6a2f2929f doc_id: 835374 cord_uid: 7z6ft3jh An effective vaccine is needed to halt the spread of the SARS-CoV-2 pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase 1/2 COVID-19 vaccine trial with BNT162b1, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Here we present antibody and T cell responses after BNT162b1 vaccination from a second, non-randomized open-label phase 1/2 trial in healthy adults, 18-55 years of age. Two doses of 1 to 50 g of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those in a COVID-19 convalescent human serum panel (HCS). Day 43 SARS-CoV-2 serum neutralising geometric mean titers were 0.7-fold (1 g) to 3.5-fold (50 g) those of HCS. Immune sera broadly neutralised pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had TH1 skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon (IFN){gamma} was produced by a high fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T-cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest multiple beneficial mechanisms with potential to protect against COVID-19. Of note, although at 1 µg BNT162b1 the immunogenicity rate was lower (6/8 responding participants), 159 the magnitude of vaccine-induced CD4 + and CD8 + T cells in some participants was almost as high as 160 with 50 µg BNT162b1 (Figure 3a ). To assess functionality and polarisation of RBD-specific T cells, 161 cytokines secreted in response to stimulation with overlapping peptides representing the full length 162 sequence of the vaccine encoded RBD were determined by intracellular staining (ICS) for IFNγ, IL-2 163 and IL-4 specific responses in pre-and post-vaccination PBMCs of 18 BNT162b1 immunised 164 participants. RBD-specific CD4 + T cells secreted IFNγ, IL-2, or both, but did not secrete IL-4 ( Figure 4 165 a-c). Similarly, fractions of RBD-specific CD8 + T cells secreted IFNγ + and IL-2. 166 The mean fraction of RBD-specific T cells within total circulating T cells obtained by BNT162b1 167 vaccination was substantially higher than that observed in six participants who recovered from COVID-168 19. Fractions of RBD-specific IFNγ + CD8 + T cells reached up to several percent of total peripheral blood 169 CD8 + T cells (Figure 4c ). Analysis of supernatants of PBMCs stimulated ex vivo with overlapping RBD 170 peptides from a subgroup of five vaccinated participants detected proinflammatory cytokines TNF, IL-In summary, these findings indicate that BNT162b1 induces functional and proinflammatory 173 CD4 + /CD8 + T cell responses in almost all participants, with TH1 polarisation of the helper response. 174 175 . 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. (which was not certified by peer review) The copyright holder for this preprint this version posted July 20, 2020. . https://doi.org/10.1101/2020.07.17.20140533 doi: medRxiv preprint CD8 + T cells, and robust release of immune-modulatory cytokines such as IFNγ, which represents a 178 coordinated immune response to counter a viral intrusion (for review 24 ). IFNγ is a key cytokine for 179 several antiviral responses. It acts in synergy with type I interferons to inhibit replication of SARS-CoV-180 2 25 . Patients with IFNγ gene polymorphism related to impaired IFNγ activity have been shown to display 181 5-fold increased susceptibility to SARS 26 . The robust production of IFNγ from CD8 + T cells indicates a 182 favourable immune response with both anti-viral and immune-augmenting properties. 183 The detection of IFNγ, IL-2 and IL-12p70 but not IL-4 indicates a favorable TH1 profile and the absence 184 of a potentially deleterious TH2 immune response. CD4 + and CD8 + T cells may confer long-lasting 185 immunity against corona viruses as indicated in SARS-CoV-1 survivors, where CD8 + T-cell immunity 186 persisted for 6-11 years 24, 27 . The study confirms the dose-dependency of RBD-binding IgG and neutralisation responses and 196 reproduces our previous findings for the 10 and 30 µg dose levels of BNT162b1 in the US trial. 197 A notable observation is that two injections of BNT162b1 at a dose level as low as 1 µg are capable of 198 inducing RBD-binding IgG levels higher than those observed in convalescent sera, and serum 199 neutralising antibody titers that were still increasing up to Day 43. Considering that it is not known 200 which neutralising antibody titer would be protective, and given the substantial T-cell responses we 201 observed for some participants in the 1 µg cohort, a considerable fraction of individuals may benefit 202 even from this lowest tested dose level. 203 A purely RBD-directed immunity might be considered prone to escape of the virus by single amino acid 204 changes in this small domain. To address this concern, neutralisation assays were conducted with 17 205 pseudo-typed viruses, 16 of which enter cells using a spike with a different RBD variant found in 206 circulating strains and one of which uses the dominant spike variant D614G. All 17 variants were 207 efficiently neutralised by BNT162b1 immune sera. 208 Limitations of our clinical study include the small sample size and its restriction to participants below 209 55 years of age. Another constraint is that we did not perform further T cell analysis e.g. deconvolution 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 July 20, 2020. . https://doi.org/10.1101/2020.07.17.20140533 doi: medRxiv preprint of epitope diversity, characterisation of HLA restriction and TCR repertoire analysis before and after 211 vaccination, due to the limited blood volumes that were available for biomarker analyses. Further, as 212 vaccine-induced immunity can wane over time, it is important to study persistence of potentially 213 protective immune responses over time. However, samples to assess persistence are not yet available 214 but are planned per study protocol and will be reported elsewhere. 215 . 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 July 20, 2020. BioNTech is the Sponsor of the study and responsible for the design, data collection, data analysis, data 253 interpretation, and writing of the report. Pfizer advised on the study and the manuscript, generated 254 serological data, and contracted for the generation of serological data. The corresponding authors had 255 full access to all the data in the study and had final responsibility for the decision to submit the data for 256 publication. All study data were available to all authors. This study was not supported by any external 257 funding at the time of submission. 258 Supplementary Information is available for this paper. 260 Correspondence and requests for materials should be addressed to Ugur Sahin. 261 . 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 July 20, 2020. The data that support the findings of this study are available from the corresponding author upon 438 reasonable request. Upon completion of this clinical trial, summary-level results will be made public 439 and shared in line with data sharing guidelines. 440 . 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 July 20, 2020. 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 July 20, 2020. . 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 July 20, 2020. . 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 July 20, 2020. Nonparametric Spearman correlation. . 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 July 20, 2020. The vaccination schedule and PBMC sampling are described in Figure 3 . PBMCs of vaccinees and COVID-19 recovered donors (HCS n=6; in (c)) were stimulated over night with an overlapping peptide pool representing the vaccine-encoded RBD and analysed by flow cytometry (a-c) and bead-based immunoassay (d). a, Exemplary pseudocolor flow cytometry plots of cytokine-producing CD4 + and CD8 + T cells from a 10-µg cohort participant. b, RBD-specific CD4 + T cells producing the indicated cytokine as a fraction of total cytokine-producing RBDspecific CD4 + T cells. c, RBD-specific CD8 + (left) or CD4 + (right) T cells producing the indicated cytokine as a fraction of total circulating T cells of the same subset. One CD4 non-responder (<0.02%total cytokine producing T cells) from the 30-µg cohort was excluded in (b). Values above data points are the mean fractions across all dose cohorts. d, PBMCs from the 50-µg cohort. Each dot represents the mean from duplicate wells subtracted by the DMSO control for one study participant. Lower limits of quantification (LLOQ) were 6.3 pg/mL for TNF, 2.5 pg/mL for IL-1β, 7.6 pg/mL for IL-12p70, 11.4 pg/mL for IL-4 and 5.3 pg/mL for IL-5. Mean (b). . 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 July 20, 2020. . . 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 July 20, 2020. Days 1 (all dose levels) and 22 (all dose levels except 60 µg) (n=12 per group; n=11 for 10 µg and 50 µg cohort from Day 22 on; discontinuation of patients due to non-vaccine related reasons; missing data points are indicated). As per protocol AEs were recorded up to 7 days after each immunisation (Days 1-7 and 22-28), and for some participants 1-2 additional days of follow-up were available. Grading of adverse events was performed according to FDA recommendations 33 . . 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 July 20, 2020. . 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 July 20, 2020. . 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. 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