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BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) that encodes the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy in preventing coronavirus disease-19 (COVID-19)1. Here we extend our previous phase 1/2 trial report2 and present BNT162b2 prime/boost induced immune response data from a second phase 1/2 trial in healthy adults (18-55 years of age). BNT162b2 elicited strong antibody responses, with SARS-CoV-2 serum 50% neutralizing geometric mean titers up to 3.3-fold above those observed in COVID-19 human convalescent samples (HCS) one week post-boost. BNT162b2-elicited sera neutralized 22 pseudoviruses bearing SARS-CoV-2 S variants. Most participants had a strong IFNγ- or IL-2-positive CD8+ and CD4+ T helper type 1 (TH1) T cell response, detectable throughout the full observation period of nine weeks following the boost. pMHC multimer technology identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week post-boost, epitope-specific CD8+ T cells of the early differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes conserved in a broad range of variants at well tolerated doses.

The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications. Keywords: double-stranded RNA, in vitro transcription, messenger RNA, nucleoside-modified RNA, RNA purification, cellulose-based purification, RNA immunogenicity

Genome editing using site-specific endonucleases (SSE) holds great potential to repair disease-causing genes. In this study we offer a promising approach to deliver SSEs as nuclease-encoded, chemically modified mRNA (nec-mRNA) together with a repair template to correct the lung disease Surfactant Protein-B (SP-B) deficiency while simultaneously reducing SSE-induced off-target effects. To assess the potential of mRNA-encoded nucleases to create site specific double-strand breaks (DSBs), we evaluated a panel of 15 transcription activator-like effector nucleases (TALENs) and 36 Zink-Finger nucleases (ZFNs) for non-homologous end-joining (NHEJ) and also tested for the integration of a promoter template by homology-directed repair (HDR) in vitro . In addition we compared different mRNA modifications to assess immunoreactions against nec-mRNA. Finally, we co-administered intratracheally nec-mRNA with the repair template to SP-B deficient mice and evaluated the survival and promoter integration efficacy. Compared to plasmid-DNA, delivery of mRNA-encoded nucleases resulted in more frequent induction of DSBs ( P P in vitro . While being only transiently expressed, incorporation of modified nucleosides into nec-mRNA and complexation into nanoparticles (NPs) increased mRNA expression levels ( P in vivo . Also, no immune activation was observed. Importantly, delivery of nec-mRNA-NP to SP-B deficient mice lead to prolonged survival rates compared with matched control groups ( P In conclusion, we have shown that delivery of nec-mRNA-NP together with a repair template results in successful, site-specific genome editing in vivo .

In vitro-transcribed (IVT) mRNA encoding therapeutic protein has the potential to treat a variety of diseases by serving as template for translation in the patient. To optimize conditions for such therapy, reporter protein-encoding mRNAs are usually used. One preferred reporter is erythropoietin (EPO), which stimulates erythropoiesis and leads to an increase in hematocrit. Measurement of hematocrit is a fast and reliable method to determine the potency of the in vitro-transcribed EPO mRNA. However, frequent blood draw from mice can increase hematocrit due to blood loss. Therefore, instead of using conventional hematocrit capillary tubes, we adapted glass microcapillaries for hematocrit measurement. Daily monitoring of mice can be accomplished by drawing less than 20 μL of blood, thus avoiding blood loss-related hematocrit increase. Due to the small volume of the withdrawn blood the hematocrit remains the same for mice injected with control mRNA, whereas significant hematocrit increase is measured between day 4 and 20 postinjection for those injected with pseudouridine-modified EPO mRNA. Following hematocrit measurement the microcapillaries are snapped easily to recover plasma for further analyses, including EPO measurement by ELISA.

Apart from conventional CD4(+) Th17 cells, the cytokines IL-17A and IL-22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T-cell receptor stimulation in the presence of TGF-β, IL-6 and IL-23. In contrast, a subset of γδ T cells ("γδT17") is committed for fast IL-17 production already in the thymus; however, γδ T cells can also produce IL-17 after prolonged in vitro stimulation via their γδ T-cell receptor plus IL-23. Here, we show that γδ T-, LTi- and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL-17A and IL-22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL-17A-producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4(-/-)) mice during experimental autoimmune encephalomyelitis. IL-17A-producing WT and Irf4(-/-) γδ T cells equally express CCR6 and lack CD27. The underlying IRF4-independent pathway partially involves STAT3 during in vitro stimulation.

Th1- and Th2-polarized immune responses are crucial in the defense against pathogens but can also promote autoimmunity and allergy. The chemokine receptors CXCR3 and CCR4 have been implicated in differential trafficking of IFN-γ- and IL-4-producing T cells, respectively, but also in tissue and inflammation-specific homing independent of cytokine responses. Here, we tested whether CD4+ T cells isolated from murine tissues under homeostatic or inflammatory conditions exhibit restricted patterns of chemotactic responses that correlate with their production of IFN-γ, IL-4, or IL-10. In uninfected mice, IL-4-producing T cells preferentially migrated to the CCR4 ligand, CCL17, whereas IFN-γ-expressing T cells as well as populations of IL-4+ or IL-10+ T cells migrated to the CXCR3 ligand, CXCL9. All cytokine-producing T cell subsets strongly migrated to the CXCR4 ligand, CXCL12. We assessed chemotaxis of T cells isolated from mice infected with influenza A virus or the nematode Nippostrongylus brasiliensis, which induce a strong Th1 or Th2 response in the lung, respectively. Unexpectedly, the chemotactic responses of IL-4+ T cells and T cells expressing the immunosuppressive cytokine IL-10 were influenced not only by the strongly Th1- or Th2-polarized environments but also by their anatomical localization, i.e., lung or spleen. In contrast, IFN-γ+ T cells exhibited robust chemotaxis toward CXCL9 and had the most consistent migration pattern in both infection models. The results support a model in which the trafficking responses of many effector and regulatory T cells are regulated as a function of the infectious and tissue environments.

Apart from conventional CD4(+) Th17 cells, the cytokines IL-17A and IL-22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T-cell receptor stimulation in the presence of TGF-β, IL-6 and IL-23. In contrast, a subset of γδ T cells ("γδT17") is committed for fast IL-17 production already in the thymus; however, γδ T cells can also produce IL-17 after prolonged in vitro stimulation via their γδ T-cell receptor plus IL-23. Here, we show that γδ T-, LTi- and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL-17A and IL-22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL-17A-producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4(-/-)) mice during experimental autoimmune encephalomyelitis. IL-17A-producing WT and Irf4(-/-) γδ T cells equally express CCR6 and lack CD27. The underlying IRF4-independent pathway partially involves STAT3 during in vitro stimulation.

Differentiation of murine T-helper (Th) 17 cells is induced by antigenic stimulation and the sequential action of the cytokines IL-6, IL-21, and IL-23, along with TGFβ. Current dogma proposes that IL-6 induces IL-21, which, in a STAT3-dependent manner, amplifies its own transcription, contributes to IL-17 production, and, moreover, promotes the expression of the IL-23 receptor. This, in turn, prepares cells for IL-23-mediated stabilization of the Th17 phenotype. Here we demonstrate that these effects of IL-21 on Th17 differentiation are completely dependent on IFN regulatory factor 4 (IRF4). After culturing in the presence of IL-21 plus TGFβ, IRF4-deficient ( Irf4 −/− ) Th cells showed a profound intrinsic defect in IL-17 production and in the autocrine IL-21 loop. Likewise, the levels of IL-23 receptor and the lineage-specific orphan nuclear receptors RORα and RORγt were diminished, whereas the T regulatory (Treg) transcription factor forkhead box P3 (Foxp3) was strongly up-regulated, consistent with the reciprocal relationship between Th17 and Treg development. Despite this loss of IL-21 functions, IL-21-induced STAT3 activation was unimpaired and induced normal Socs3 expression. Forced expression of Foxp3 in WT cells inhibited IL-21-mediated IL-17 production, suggesting that the increase in Foxp3 contributes to the Irf4 −/− phenotype. Additionally, the low levels of RORα and RORγt are also partially responsible, because simultaneous overexpression of both proteins restored IL-17 production in Irf4 −/− cells to some extent. These data highlight IRF4 as a decisive factor during the IL-21-mediated steps of Th17 development by influencing the balance of Foxp3, RORα, and RORγt.