Assessment of Immunogenicity and Efficacy of CV0501 mRNA-Based Omicron COVID-19 Vaccination in Small Animal Models.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron and its subvariants (BA.2, BA.4, BA.5) represented the most commonly circulating variants of concern (VOC) in the coronavirus disease 2019 (COVID-19) pandemic in 2022. Despite high vaccination rates with approved SARS-CoV-2 vaccines encoding the ancestral spike (S) protein, these Omicron subvariants have collectively resulted in increased viral transmission and disease incidence. This necessitates the development and characterization of vaccines incorporating later emerging S proteins to enhance protection against VOC. In this context, bivalent vaccine formulations may induce broad protection against VOC and potential future SARS-CoV-2 variants. Here, we report preclinical data for a lipid nanoparticle (LNP)-formulated RNActive® N1-methylpseudouridine (N1mΨ) modified mRNA vaccine (CV0501) based on our second-generation SARS-CoV-2 vaccine CV2CoV, encoding the S protein of Omicron BA.1. The immunogenicity of CV0501, alone or in combination with a corresponding vaccine encoding the ancestral S protein (ancestral N1mΨ), was first measured in dose-response and booster immunization studies performed in Wistar rats. Both monovalent CV0501 and bivalent CV0501/ancestral N1mΨ immunization induced robust neutralizing antibody titers against the BA.1, BA.2 and BA.5 Omicron subvariants, in addition to other SARS-CoV-2 variants in a booster immunization study. The protective efficacy of monovalent CV0501 against live SARS-CoV-2 BA.2 infection was then assessed in hamsters. Monovalent CV0501 significantly reduced SARS-CoV-2 BA.2 viral loads in the airways, demonstrating protection induced by CV0501 vaccination. CV0501 has now advanced into human Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT05477186).

Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models.

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve control of the COVID-19 pandemic. We compare monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein in a transgenic mouse and a Wistar rat model. The blended low-dose bivalent mRNA vaccine contains half the mRNA of each respective monovalent vaccine, but induces comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, antigen-specific CD4+ and CD8+ responses, and protects transgenic female mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduces viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized female Wistar rats also contain neutralizing antibodies against the B.1.1.529 (Omicron BA.1 and BA.5) variants. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins are feasible approaches for extending the coverage of vaccines for emerging and co-circulating SARS-CoV-2 variants.

A third dose of the unmodified COVID-19 mRNA vaccine CVnCoV enhances quality and quantity of immune responses.

A third vaccine dose is often required to achieve potent, long-lasting immune responses. We investigated the effect of three 8-µg doses of CVnCoV, CureVac's severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate containing sequence-optimized unmodified mRNA encoding the spike (S) glycoprotein, administered at 0, 4, and 28 weeks, on immune responses in rhesus macaques. After the third dose, S-specific binding and neutralizing antibodies increased 50-fold compared with post-dose 2 levels, with increased responses also evident in the lower airways and against the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants. Enhanced binding affinity of serum antibodies after the third dose correlated with higher somatic hypermutation in S-specific B cells, corresponding with improved binding properties of monoclonal antibodies expressed from isolated B cells. Administration of low-dose mRNA led to fewer cells expressing antigen in vivo at the injection site and in the draining lymph nodes compared with a 10-fold higher dose, possibly reducing engagement of precursor cells with the antigen and resulting in the suboptimal response observed after two-dose vaccination schedules in phase IIb/III clinical trials of CVnCoV. However, when immune memory is established, a third dose efficiently boosts the immunological responses and improves antibody affinity and breadth.

Optimised Non-Coding Regions of mRNA SARS-CoV-2 Vaccine CV2CoV Improves Homologous and Heterologous Neutralising Antibody Responses.

More than two years after the emergence of SARS-CoV-2, 33 COVID-19 vaccines, based on different platforms, have been approved in 197 countries. Novel variants that are less efficiently neutralised by antibodies raised against ancestral SARS-CoV-2 are circulating, highlighting the need to adapt vaccination strategies. Here, we compare the immunogenicity of a first-generation mRNA vaccine candidate, CVnCoV, with a second-generation mRNA vaccine candidate, CV2CoV, in rats. Higher levels of spike (S) protein expression were observed in cell culture with the CV2CoV mRNA than with the CVnCoV mRNA. Vaccination with CV2CoV also induced higher titres of virus neutralising antibodies with accelerated kinetics in rats compared with CVnCoV. Significant cross-neutralisation of the SARS-CoV-2 variants, Alpha (B.1.1.7), Beta (B.1.351), and the 'mink' variant (B1.1.298) that were circulating at the time in early 2021 were also demonstrated. In addition, CV2CoV induced higher levels of antibodies at lower doses than CVnCoV, suggesting that dose-sparing could be possible with the next-generation SARS-CoV-2 vaccine, which could improve worldwide vaccine supply.

mRNA vaccines induce rapid antibody responses in mice.

mRNA vaccines can be developed and produced quickly, making them prime candidates for immediate outbreak responses. Furthermore, clinical trials have demonstrated rapid protection following mRNA vaccination. Thus, we sought to investigate how quickly mRNA vaccines elicit antibody responses compared to other vaccine modalities. We first compared the immune kinetics of mRNA and DNA vaccines expressing SARS-CoV-2 spike in mice. We observed rapid induction of antigen-specific binding and neutralizing antibodies by day 5 following mRNA (4 µg/mouse), but not DNA (50 µg/mouse), immunization. Comparing innate responses hours post immunization, the mRNA vaccine induced increased levels of IL-5, IL-6, and MCP-1 cytokines which maybe promoting humoral responses downstream. We then evaluated the immune kinetics of an HIV-1 mRNA vaccine in comparison to DNA, protein, and rhesus adenovirus 52 (RhAd52) vaccines of the same HIV-1 envelope antigen in mice. Again, induction of envelope-specific antibodies was observed by day 5 following mRNA vaccination, whereas antibodies were detected by day 7-14 following DNA, protein, and RhAd52 vaccination. Thus, eliciting rapid humoral immunity may be a unique and advantageous property of mRNA vaccines for controlling infectious disease outbreaks.

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine.

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

mRNA Vaccines Induce Rapid Antibody Responses in Mice.

mRNA vaccines can be developed and produced quickly, making them attractive for immediate outbreak responses. Furthermore, clinical trials have demonstrated rapid protection following mRNA vaccination. We sought to investigate how quickly mRNA vaccines elicit antibody responses compared to other vaccine modalities. We first examined immune kinetics of mRNA and DNA vaccines expressing SARS-CoV-2 spike in mice. We observed rapid induction of antigen-specific binding and neutralizing antibodies by day 5 following mRNA, but not DNA, immunization. The mRNA vaccine also induced increased levels of IL-5, IL-6 and MCP-1. We then evaluated immune kinetics of an HIV-1 mRNA vaccine in comparison to DNA, protein, and rhesus adenovirus 52 (RhAd52) vaccines with the same HIV-1 envelope antigen in mice. Induction of envelope-specific antibodies was observed by day 5 following mRNA vaccination, whereas antibodies were detected by day 7-14 following DNA, protein, and RhAd52 vaccination. Eliciting rapid humoral immunity may be an advantageous property of mRNA vaccines for controlling infectious disease outbreaks. IMPORTANCE: mRNA vaccines can be developed and produced in record time. Here we demonstrate induction of rapid antibody responses by mRNA vaccines encoding two different viral antigens by day 5 following immunization in mice. The rapid immune kinetics of mRNA vaccines can be an advantageous property that makes them well suited for rapid control of infectious disease outbreaks.

Safety and immunogenicity of an mRNA-lipid nanoparticle vaccine candidate against SARS-CoV-2 : A phase 1 randomized clinical trial.

BACKGROUND: We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV­2 spike (S) protein encapsulated in lipid nanoparticles (LNP). METHODS: This is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV­2 S­protein and receptor binding domain (RBD), and SARS-CoV­2 neutralizing titers (MN50). RESULTS: In 245 volunteers who received 2 CVnCoV vaccinations (2 µg, n = 47, 4 µg, n = 48, 6 µg, n = 46, 8 µg, n = 44, 12 µg, n = 28) or placebo (n = 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to S­protein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to S­protein or RBD, and 83% (19/23) seroconverted for MN50 in the 12 µg group. Responses to 12 µg were comparable to those observed in convalescent sera from known COVID-19 patients. CONCLUSION: In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12 µg dosages elicited levels of immune responses that overlapped those observed in convalescent sera.

CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2.

The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.

IABS/CEPI platform technology webinar: Is it possible to reduce the vaccine development time?

The International Alliance for Biological Standardization and the Coalition for Epidemic Preparedness Innovations organized a joint webinar on the use of platform technologies for vaccine development. To tackle new emerging infectious diseases, including SARS-CoV-2, rapid response platforms, using the same basic components as a backbone, yet adaptable for use against different pathogens by inserting new genetic or protein sequences, are essential. Furthermore, it is evident that development of platform technologies needs to continue, due to the emerging variants of SARS-CoV-2. The objective of the meeting was to discuss techniques for platform manufacturing that have been used for COVID-19 vaccine development, with input from regulatory authorities on their experiences with, and expectations of, the platforms. Industry and regulators have been very successful in cooperating, having completed the whole process from development to licensing at an unprecedented speed. However, we should learn from the experiences, to be able to be even faster when a next pandemic of disease X occurs.

mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents.

mRNA technologies have recently proven clinical efficacy against coronavirus disease 2019 and are among the most promising technologies to address the current pandemic. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle-encapsulated mRNA vaccine that encodes full-length, pre-fusion stabilised severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. In contrast to previously published approaches, CVnCoV is exclusively composed of naturally occurring nucleotides. Immunisation with CVnCoV induced strong humoral responses with high titres of virus-neutralising antibodies and robust T-cell responses. CVnCoV vaccination protected hamsters from challenge with wild-type SARS-CoV-2, demonstrated by the absence of viral replication in the lungs. Hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication exhibited no evidence of vaccine-enhanced disease. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.

Drug biokinetic and toxicity assessments in rat and human primary hepatocytes and HepaRG cells within the EU-funded Predict-IV project.

The overall aim of Predict-IV (EU-funded collaborative project #202222) was to develop improved testing strategies for drug safety in the late discovery phase. One major focus was the prediction of hepatotoxicity as liver remains one of the major organ leading to failure in drug development, drug withdrawal and has a poor predictivity from animal experiments. In this overview we describe the use and applicability of the three cell models employed, i.e., primary rat hepatocytes, primary human hepatocytes and the human HepaRG cell line, using four model compounds, chlorpromazine, ibuprofen, cyclosporine A and amiodarone. This overview described the data generated on mode of action of liver toxicity after long-term repeat-dosing. Moreover we have quantified parent compound and its distribution in various in vitro compartments, which allowed us to develop biokinetic models where we could derive real exposure concentrations in vitro. In conclusion, the complex data set enables quantitative measurements that proved the concept that we can define human relevant free and toxic exposure levels in vitro. Further compounds have to be analyzed in a broader concentration range to fully exploit these promising results for improved prediction of hepatotoxicity and hazard assessment for humans.

Transcriptomics hit the target: Monitoring of ligand-activated and stress response pathways for chemical testing.

High content omic methods provide a deep insight into cellular events occurring upon chemical exposure of a cell population or tissue. However, this improvement in analytic precision is not yet matched by a thorough understanding of molecular mechanisms that would allow an optimal interpretation of these biological changes. For transcriptomics (TCX), one type of molecular effects that can be assessed already is the modulation of the transcriptional activity of a transcription factor (TF). As more ChIP-seq datasets reporting genes specifically bound by a TF become publicly available for mining, the generation of target gene lists of TFs of toxicological relevance becomes possible, based on actual protein-DNA interaction and modulation of gene expression. In this study, we generated target gene signatures for Nrf2, ATF4, XBP1, p53, HIF1a, AhR and PPAR gamma and tracked TF modulation in a large collection of in vitro TCX datasets from renal and hepatic cell models exposed to clinical nephro- and hepato-toxins. The result is a global monitoring of TF modulation with great promise as a mechanistically based tool for chemical hazard identification.

Understanding the biokinetics of ibuprofen after single and repeated treatments in rat and human in vitro liver cell systems.

Common in vitro toxicity testing often neglects the fate and intracellular concentration of tested compounds, potentially limiting the predictability of in vitro results for in vivo extrapolation. We used in vitro long-term cultures of primary rat (PRH) and human hepatocytes (PHH) and HepaRG cells to characterise and model the biokinetic profile of ibuprofen (IBU) after single and daily repeated exposure (14 days) to two concentrations. A cross-model comparison was carried out at 100µM, roughly corresponding to the human therapeutic plasma concentration. Our results showed that IBU uptake was rapid and a dynamic equilibrium was reached within 1 or 2 days. All three cell systems efficiently metabolised IBU. In terms of species-differences, our data mirrored known in vivo results. Although no bioaccumulation was observed, IBU intracellular concentration was higher in PRH due to a 10-fold lower metabolic clearance compared to the human-derived cells. In HepaRG cells, IBU metabolism increased over time, but was not related to the treatment. In PHH, a low CYP2C9 activity, the major IBU-metabolising CYP, led to an increased cytotoxicity. A high inter-individual variability was seen in PHH, whereas HepaRG cells and PRH were more reproducible models. Although the concentrations of IBU in PRH over time differed from the concentrations found in human cells under similar exposure conditions.

Comprehensive summary--Predict-IV: A systems toxicology approach to improve pharmaceutical drug safety testing.

This special issue of Toxicology in Vitro is dedicated to disseminating the results of the EU-funded collaborative project "Profiling the toxicity of new drugs: a non animal-based approach integrating toxicodynamics and biokinetics" (Predict-IV; Grant 202222). The project's overall aim was to develop strategies to improve the assessment of drug safety in the early stage of development and late discovery phase, by an intelligent combination of non animal-based test systems, cell biology, mechanistic toxicology and in silico modeling, in a rapid and cost effective manner. This overview introduces the scope and overall achievements of Predict-IV.

Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure.

Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14 days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1-2 µM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20 µM. Overall, the mass balance of CPZ decreased in the course of 24 h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure. Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data.

Transcriptomic hepatotoxicity signature of chlorpromazine after short- and long-term exposure in primary human sandwich cultures.

Drug-induced liver injury is the most frequent reason for market withdrawal of approved drugs, and is difficult to predict in animal models. Here, we analyzed transcriptomic data derived from short- and long-term cultured primary human hepatocytes (PHH) exposed to the well known human hepatotoxin chlorpromazine (CPZ). Samples were collected from five PHH cultures after short-term (1 and 3 days) and long-term (14 days) repeat daily treatment with 0.1 or 0.2 µM CPZ, corresponding to C(max). Two PHH cultures were additionally treated with 1 µM CPZ, and the three others with 0.02 µM CPZ. Differences in the total number of gene changes were seen between donors and throughout treatment. Specific transcriptomic hepatotoxicity signatures were created for CPZ and consisted of inflammation/hepatitis, cholestasis, and liver proliferation in all five donors, as well as fibrosis and steatosis, which were observed in four of five donors. Necrosis was present in three of five donors, and an indicative signature of cirrhosis was observed after long-term 14-day repeat treatment, also in three of five donors. The inter-donor variability in the inflammatory response to CPZ treatment was associated with variability in the strength of the response of the transcriptomic hepatotoxicity signatures, suggesting that features of inflammation could be related to the idiosyncratic hepatotoxic effects of CPZ in humans.

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress.

High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15µM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5µM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress.

A low-toxicity IL-2-based immunocytokine retains antitumor activity despite its high degree of IL-2 receptor selectivity.

PURPOSE: The goal of the study was to engineer a form of interleukin 2 (IL-2) that, when delivered as a tumor-specific antibody fusion protein, retains the ability to stimulate an antitumor immune response via interaction with the high-affinity IL-2 receptor but has lower toxicity because of the reduced activation of the intermediate-affinity IL-2 receptor. EXPERIMENTAL DESIGN: We investigated changes in the proposed toxin motif of IL-2 by introducing a D20T mutation that has little effect on the activity of free IL-2. We expressed this IL-2 variant as a fusion protein with an antibody (NHS76) that targets the necrotic core of tumors and characterized this molecule (NHS-IL2LT) in vitro and in vivo. RESULTS: NHS-IL2LT was shown to have near normal biological activity in vitro by using T-cell lines expressing the high-affinity IL-2 receptor, but little or no activity by using cell lines expressing only the intermediate IL-2 receptor. Relative to the control antibody fusion protein containing wild-type IL-2, NHS-IL2LT retained antitumor activity against established neuroblastoma and non-small cell lung cancer metastases in syngeneic mouse tumor models but was much better tolerated in immune-competent mice and in cynomolgus monkeys. CONCLUSIONS: The qualities of low toxicity and single-agent efficacy shown suggest that NHS-IL2LT is a good candidate for therapeutic approaches combining standard cytotoxic and immune therapies. In fact, this molecule (also known as Selectikine or EMD 521873) is currently in phase I clinical trial.

Genomic profiling uncovers a molecular pattern for toxicological characterization of mutagens and promutagens in vitro.

The aim of this study was to evaluate the suitability of global gene expression profiling for the characterization and identification of mutagens and promutagens in vitro. To enable detection of both mutagenic and promutagenic compounds, we cotreated HepG2 cells with a rat liver S9 fraction as metabolic activation system (MAS), supplementing the limited drug metabolic capability of HepG2 cells. Illumina BeadChip arrays were used to quantify gene expression changes after treatment with three well-known mutagenic, three promutagenic, as well as two non-genotoxic reference compounds for a period of 24 or 48 h. Statistical data analysis revealed 91 genes being most representative for the (pro-)genotoxic response. Several processes such as cellular differentiation and the complex interactive regulation of the stress and DNA damage response via the transcriptional modulators STAT1, SP1, and P53 were differentially regulated. The gene set evaluated was further used to predict the genotoxic characteristics of N-nitrosodiethylamine (DEN) after its metabolic activation. Although no clear response could be established in P53 activation experiments, DEN was classified correctly as nongenotoxic without S9 and genotoxic in the presence of the MAS by means of its transcriptomic pattern. Our data support that mechanistic profiling in vitro is a useful tool compared with single endpoint detections to predict genotoxicity.