The Discovery of Novel α2a Adrenergic Receptor Agonists Only Coupling to Gαi/O Proteins by Virtual Screening.

Most α2-AR agonists derived from dexmedetomidine have few structural differences between them and have no selectivity for α2A/2B-AR or Gi/Gs, which can lead to side effects in drugs. To obtain novel and potent α2A-AR agonists, we performed virtual screening for human α2A-AR and α2B-AR to find α2A-AR agonists with higher selectivity. Compound P300-2342 and its three analogs significantly decreased the locomotor activity of mice (p < 0.05). Furthermore, P300-2342 and its three analogs inhibited the binding of [3H] Rauwolscine with IC50 values of 7.72 ± 0.76 and 12.23 ± 0.11 µM, respectively, to α2A-AR and α2B-AR. In α2A-AR-HEK293 cells, P300-2342 decreased forskolin-stimulated cAMP production without increasing cAMP production, which indicated that P300-2342 activated α2A-AR with coupling to the Gαi/o pathway but without Gαs coupling. P300-2342 exhibited no agonist but slight antagonist activities in α2B-AR. Similar results were obtained for the analogs of P300-2342. The docking results showed that P300-2342 formed π-hydrogen bonds with Y394, V114 in α2A-AR, and V93 in α2B-AR. Three analogs of P300-2342 formed several π-hydrogen bonds with V114, Y196, F390 in α2A-AR, and V93 in α2B-AR. We believe that these molecules can serve as leads for the further optimization of α2A-AR agonists with potentially few side effects.

Design optimization of geometrically confined cardiac organoids enabled by machine learning techniques.

Stem cell organoids are powerful models for studying organ development, disease modeling, drug screening, and regenerative medicine applications. The convergence of organoid technology, tissue engineering, and artificial intelligence (AI) could potentially enhance our understanding of the design principles for organoid engineering. In this study, we utilized micropatterning techniques to create a designer library of 230 cardiac organoids with 7 geometric designs. We employed manifold learning techniques to analyze single organoid heterogeneity based on 10 physiological parameters. We clustered and refined the cardiac organoids based on their functional similarity using unsupervised machine learning approaches, thus elucidating unique functionalities associated with geometric designs. We also highlighted the critical role of calcium transient rising time in distinguishing organoids based on geometric patterns and clustering results. This integration of organoid engineering and machine learning enhances our understanding of structure-function relationships in cardiac organoids, paving the way for more controlled and optimized organoid design.

An ab initio molecular dynamics investigation of the behaviour of amorphous substances in anodic aluminium oxide under electric field.

In order to elucidate the diffusion behaviour of ions in alumina during the anodic alumina process, the effects of electric field strength, hydration content, and electrolyte on amorphous alumina and hydrated alumina were studied using ab initio molecular dynamics. The results show that the diffusion rate of ions in alumina increases with the increase in electric field strength, but there is an extreme value. The maximum diffusion rate of Al ions in alumina monohydrate is 21.8 µm2/ms/V, while in alumina trihydrate, it is 16.7 µm2/ms/V. The ionic diffusion rate of hydrated alumina is one to two orders of magnitude larger than that of anhydrous amorphous alumina due to the effect of the drag of H ions, which reduces the migration activation energy. Electrolytes also affect the diffusion rate of alumina through the action of H ions. The increase in H ions will not only enhance the diffusion rate of hydrated alumina but also render the hydrous compound more vulnerable to breakdown.

Exploration and structure-activity relationship research of benzenesulfonamide derivatives as potent TRPV4 inhibitors for treating acute lung injury.

RN-9893, a TRPV4 antagonist identified by Renovis Inc., showcased notable inhibition of TRPV4 channels. This research involved synthesizing and evaluating three series of RN-9893 analogues for their TRPV4 inhibitory efficacy. Notably, compounds 1b and 1f displayed a 2.9 to 4.5-fold increase in inhibitory potency against TRPV4 (IC50 = 0.71 ± 0.21 µM and 0.46 ± 0.08 µM, respectively) in vitro, in comparison to RN-9893 (IC50 = 2.07 ± 0.90 µM). Both compounds also significantly outperformed RN-9893 in TRPV4 current inhibition rates (87.6 % and 83.2 % at 10 µM, against RN-9893's 49.4 %). For the first time, these RN-9893 analogues were profiled in an in vivo mouse model, where intraperitoneal injections of 1b or 1f at 10 mg/kg notably mitigated symptoms of acute lung injury induced by lipopolysaccharide (LPS). These outcomes indicate that compounds 1b and 1f are promising candidates for acute lung injury treatment.

An investigation using DFT into the impact of hydrogen on oxygen migration processes during aluminum anodization.

First-principles computations were utilized to examine the impact of H atoms on the surface behavior of O atoms on the (111) surface of Al and their infiltration behavior into the Al crystal, with the aim of elucidating the behavior of ions in the anodic process during aluminum oxidation. According to the findings, the "abstract" action of H atoms significantly lowers the energy barrier preventing O from entering the Al crystal. The addition of a H atom influences the diffusion of O atoms in the Al crystal as well, and this can lower the activation energy of O atom migration between the tetrahedral interstitial locations from 1.23 eV to 0.35 eV. We can benefit from knowing how ions are transported and anodic oxidation occurs.

Novel Scaffold Agonists of the α2A Adrenergic Receptor Identified via Ensemble-Based Strategy.

The α2A adrenergic receptor (α2A-AR) serves as a critical molecular target for sedatives and analgesics. However, α2A-AR ligands with an imidazole ring also interact with an imidazoline receptor as well as other proteins and lead to undesirable effects, motivating us to develop more novel scaffold α2A-AR ligands. For this purpose, we employed an ensemble-based ligand discovery strategy, integrating long-term molecular dynamics (MD) simulations and virtual screening, to identify new potential α2A-AR agonists with novel scaffold. Our results showed that compounds SY-15 and SY-17 exhibited significant biological effects in the preliminary evaluation of protein kinase A (PKA) redistribution assays. They also reduced levels of intracellular cyclic adenosine monophosphate (cAMP) in a dose-dependent manner. Upon treatment of the cells with 100 µM concentrations of SY-15 and SY-17, there was a respective decrease in the intracellular cAMP levels by 63.43% and 53.83%. Subsequent computational analysis was conducted to elucidate the binding interactions of SY-15 and SY-17 with the α2A-AR. The binding free energies of SY-15 and SY-17 calculated by MD simulations were -45.93 and -71.97 kcal/mol. MD simulations also revealed that both compounds act as bitopic agonists, occupying the orthosteric site and a novel exosite of the receptor simultaneously. Our findings of integrative computational and experimental approaches could offer the potential to enhance ligand affinity and selectivity through dual-site occupancy and provide a novel direction for the rational design of sedatives and analgesics.

Thermally and Photothermally Triggered Cytocompatible Triple-Shape-Memory Polymer Based on a Graphene Oxide-Containing Poly(ε-caprolactone) and Acrylate Composite.

Triple-shape-memory polymers (triple-SMPs) are a class of polymers capable of fixing two temporary shapes and recovering sequentially from the first temporary shape to the second temporary shape and, last, to the permanent shape. To accomplish a sequential shape change, a triple-SMP must have two separate shape-fixing mechanisms triggerable by distinct stimuli. Despite the biomedical potential of triple-SMPs, a triple-SMP that with cells present can undergo two different shape changes via two distinct cytocompatible triggers has not previously been demonstrated. Here, we report the design and characterization of a cytocompatible triple-SMP material that responds separately to thermal and light triggers to undergo two distinct shape changes under cytocompatible conditions. Tandem triggering was achieved via a photothermally triggered component, comprising poly(ε-caprolactone) (PCL) fibers with graphene oxide (GO) particles physically attached, embedded in a thermally triggered component, comprising a tert-butyl acrylate-butyl acrylate (tBA-BA) matrix. The material was characterized in terms of thermal properties, surface morphology, shape-memory performance, and cytocompatibility during shape change. Collectively, the results demonstrate cytocompatible triple-shape behavior with a relatively larger thermal shape change (an average of 20.4 ± 4.2% strain recovered for all PCL-containing groups) followed by a smaller photothermal shape change (an average of 3.5 ± 0.8% strain recovered for all PCL-GO-containing groups; samples without GO showed no recovery) with greater than 95% cell viability on the triple-SMP materials, establishing the feasibility of triple-shape memory to be incorporated into biomedical devices and strategies.

Discovery of highly potent and selective KRASG12C degraders by VHL-recruiting PROTACs for the treatment of tumors with KRASG12C-Mutation.

Although several covalent KRASG12C inhibitors have made great progress in the treatment of KRASG12C-mutant cancer, their clinical applications are limited by adaptive resistance, motivating novel therapeutic strategies. Through drug design and structure optimization, a series of highly potent and selective KRASG12C Proteolysis Targeting Chimeras (PROTACs) were developed by incorporating AMG510 and VHL ligand VH032. Among them, degrader YN14 significantly inhibited KRASG12C-dependent cancer cells growth with nanomolar IC50 and DC50 values, and ＞ 95 % maximum degradation (Dmax). Molecular dynamics (MD) simulation showed that YN14 induced a stable KRASG12C: YN14: VHL ternary complex with low binding free energy (ΔG). Notably, YN14 led to tumor regression with tumor growth inhibition (TGI%) rates more than 100 % in the MIA PaCa-2 xenograft model with well-tolerated dose-schedules. We also found that KRASG12C degradation exhibited advantages in overcoming adaptive KRASG12C feedback resistance over KRASG12C inhibition. Furthermore, combination of RTKs, SHP2, or CDK9 inhibitors with YN14 exhibited synergetic efficacy in KRASG12C-mutant cancer cells. Overall, these results demonstrated that YN14 holds exciting prospects for the treatment of tumors with KRASG12C-mutation and boosted efficacy could be achieved for greater clinical applications via drug combination.

Discovery of novel exceptionally potent and orally active c-MET PROTACs for the treatment of tumors with MET alterations.

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC50 values and achieved picomolar DC50 values and >99% of maximum degradation (Dmax) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-METY1230H and c-METD1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.

Controlling Morphology and Functions of Cardiac Organoids by Two-Dimensional Geometrical Templates.

Traditionally, tissue-specific organoids are generated as 3D aggregates of stem cells embedded in Matrigel or hydrogels, and the aggregates eventually end up a spherical shape and suspended in the matrix. Lack of geometrical control of organoid formation makes these spherical organoids limited for modeling the tissues with complex shapes. To address this challenge, we developed a new method to generate 3D spatial-organized cardiac organoids from 2D micropatterned human induced pluripotent stem cell (hiPSC) colonies, instead of directly from 3D stem cell aggregates. This new approach opens the possibility to create cardiac organoids that are templated by 2D non-spherical geometries, which potentially provides us a deeper understanding of biophysical controls on developmental organogenesis. Here, we designed 2D geometrical templates with quadrilateral shapes and pentagram shapes that had same total area but different geometrical shapes. Using this templated substrate, we grew cardiac organoids from hiPSCs and collected a series of parameters to characterize morphological and functional properties of the cardiac organoids. In quadrilateral templates, we found that increasing the aspect ratio impaired cardiac tissue 3D self-assembly, but the elongated geometry improved the cardiac contractile functions. However, in pentagram templates, cardiac organoid structure and function were optimized with a specific geometry of an ideal star shape. This study will shed a light on "organogenesis-by-design" by increasing the intricacy of starting templates from external geometrical cues to improve the organoid morphogenesis and functionality.

Inhibition of cannabinoid receptor type 1 sensitizes triple-negative breast cancer cells to ferroptosis via regulating fatty acid metabolism.

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer that displays highly aggressive with poor prognosis. Owing to the limited targets and drugs for TNBC clinical therapy, it is necessary to investigate the factors regulating cancer progression and develop novel therapies for cancer treatment. Ferroptosis, a nonapoptotic form of programmed cell death characterized by accumulation of iron-dependent peroxidation of phospholipids, is regulated by cellular metabolism, redox homeostasis, and various cancer-related signaling pathways. Recently, considerable progress has been made in demonstrating the critical role of lipid metabolism in regulating ferroptosis, indicating potential combinational therapeutic strategies for cancer treatment. In this study, by drug combination screen of lipid metabolism compounds with ferroptosis inducers in decreasing TNBC cell viability, we found potent synergy of the CB1 antagonist rimonabant with erastin/(1 S, 3 R)-RSL3 (RSL3) in inhibiting TNBC cell growth both in vitro and in vivo via promoting the levels of lipid peroxides, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and cytosolic reactive oxygen species (ROS) production, enhancing intracellular glutathione (GSH) depletion and inducing G1 cell cycle arrest. We identified that inhibition of CB1 promoted the effect of erastin/RSL3 on inducing ferroptosis and enhanced their inhibitory effect on tumor growth. Using RNA-Seq, fatty acid analyses and functional assays, we found that CB1 regulated stearoyl-CoA desaturase 1 (SCD1)- and fatty acyl desaturase 2 (FADS2)-dependent fatty acid metabolism via phosphatidylinositol 3 kinase (PI3K)-AKT and mitogen-activated protein kinase (MAPK) signaling pathways to modulate ferroptosis sensitivity in TNBC cells. These data demonstrate that dual targeting of CB1 and ferroptosis could be a promising therapeutic strategy for TNBC.

Discovery of highly potent and selective CRBN-recruiting EGFRL858R/T790M degraders in vivo.

Currently, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are widely used in the treatment of non-small cell lung cancer (NSCLC). However, the inevitable drug resistance and side effects are the current main obstacle, which motivating novel therapies. Proteolysis targeting chimera (PROTAC), a lately-developed technology to target proteins for degradation, has been utilized for drug development. Therefore, we designed, synthesized and evaluated a series of CRBN-recruiting EGFR degraders. Among them, 13a and 13b significantly inhibited NCI-H1975 cells proliferation with IC50 values of 58.08 nM and 46.82 nM, respectively, whereas exhibited more than 100 µM against A549 or H1299 cells, whose selectivity was more than 1700-fold. 13a and 13b potently induced the EGFRL858R/T790M degradation by ubiquitin proteasome system in a time- and dose-dependent manner but not that of EGFRWT, and the DC50 values of 13b was 13.2 nM, which was the most potent compound in current known CRBN-recruiting EGFRL858R/T790M degraders. 13a and 13b dramatically induced cell apoptosis, cell cycle arrest and inhibited downstream signaling pathways. Furthermore, 13a and 13b effectively and selectively inhibited NCI-H1975 xenograft tumor growth with good pharmacokinetics (PK) properties in vivo. These findings demonstrate that 13a and 13b could serve as candidates for developing the drug for treating NSCLC.

Controlling Mesenchyme Tissue Remodeling via Spatial Arrangement of Mechanical Constraints.

Tissue morphogenetic remodeling plays an important role in tissue repair and homeostasis and is often governed by mechanical stresses. In this study, we integrated an in vitro mesenchymal tissue experimental model with a volumetric contraction-based computational model to investigate how geometrical designs of tissue mechanical constraints affect the tissue remodeling processes. Both experimental data and simulation results verified that the standing posts resisted the bulk contraction of the tissues, leading to tissue thinning around the posts as gap extension and inward remodeling at the edges as tissue compaction. We changed the geometrical designs for the engineered mesenchymal tissues with different shapes of posts arrangements (triangle vs. square), different side lengths (6 mm vs. 8 mm), and insertion of a center post. Both experimental data and simulation results showed similar trends of tissue morphological changes of significant increase of gap extension and deflection compaction with larger tissues. Additionally, insertion of center post changed the mechanical stress distribution within the tissues and stabilized the tissue remodeling. This experimental-computational integrated model can be considered as a promising initiative for future mechanistic understanding of the relationship between mechanical design and tissue remodeling, which could possibly provide design rationale for tissue stability and manufacturing.

Discovery of highly potent and selective EGFRT790M/L858R TKIs against NSCLC based on molecular dynamic simulation.

Epidermal growth factor receptor (EGFR) is the most attractive target for drug research in non-small cell lung cancer (NSCLC). There have been three generation drugs developed to treat of NSCLC. The third-generation EGFR tyrosine kinase inhibitors (TKIs) Rociletinib and Osimertinib (AZD9291) achieved remarkable clinical efficacy. However, due to the inhibitory activity against the wild-type EGFR, the side effect of associated skin rash and gastrointestinal toxicity appeared. Thus, there is still an urgent need to develop novel inhibitors with potent inhibitory activity and high selectivity for T790M-containing EGFR over EGFRWT. Herein, guided by the molecular dynamic simulation results, a series of potent and selective Osimertinib derivatives were designed, synthesized and evaluated. The promising compounds 7f, 7g, 7k, 7m and 7n demonstrated excellent kinase inhibitory activity and high selectivity for EGFRT790M/L858R mutant. The selectivity of 7m to EGFRT790M/L858R was the highest in the current known compounds near to 2500-fold. In addition, the compound 7m showed considerable activity against NCI-H1975 and HCC827 cells, arrested NCI-H1975 cell cycle at the G2/M stage and significantly induced apoptosis in NCI-H1975 cell. These encouraged results indicated that 7m will be used as a candidate targeting EGFRT790M/L858R for further pharmacodynamic and pharmacokinetic studies, and all these studies provide important clues for the discovery of potent EGFRT790M/L858R inhibitors with high selectivity.

Profiling the responsiveness of focal adhesions of human cardiomyocytes to extracellular dynamic nano-topography.

Focal adhesion complexes function as the mediators of cell-extracellular matrix interactions to sense and transmit the extracellular signals. Previous studies have demonstrated that cardiomyocyte focal adhesions can be modulated by surface topographic features. However, the response of focal adhesions to dynamic surface topographic changes remains underexplored. To study this dynamic responsiveness of focal adhesions, we utilized a shape memory polymer-based substrate that can produce a flat-to-wrinkle surface transition triggered by an increase of temperature. Using this dynamic culture system, we analyzed three proteins (paxillin, vinculin and zyxin) from different layers of the focal adhesion complex in response to dynamic extracellular topographic change. Hence, we quantified the dynamic profile of cardiomyocyte focal adhesion in a time-dependent manner, which provides new understanding of dynamic cardiac mechanobiology.

Remodeling of Architected Mesenchymal Microtissues Generated on Mechanical Metamaterials.

Mechanical metamaterials constitute a nascent category of architected structures comprising arranged periodic components with tailored geometrical features. These materials are now being employed as advanced medical implants due to their extraordinary mechanical properties over traditional devices. Nevertheless, to achieve desired tissue integration and regeneration, it is critical to study how the microarchitecture affects interactions between metamaterial scaffolds and living biological tissues. Based on human induced pluripotent stem cell technology and multiphoton lithography, we report the establishment of an in vitro microtissue model to study the integration and remodeling of human mesenchymal tissues on metamaterial scaffolds with different unit geometries. Microtissues showed distinct tissue morphologies and cellular behaviors between architected octet-truss and bowtie structures. Under the active force generated from mesenchymal tissues, the octet-truss and bowtie metamaterial scaffolds demonstrated unique instability phenomena, significantly different from uniform loading using conventional mechanical testing.

Combined scaffold hopping, molecular screening with dynamic simulation to screen potent CRBN ligands.

Thalidomide and its derivatives lenalidomide and pomalidomide, known as immunomodulatory drugs, (IMiDs) bind directly to cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase, resulting in the rapid ubiquitination and degradation of the substrate protein. With the discovery of the protein degradation mechanism of IMiDs, targeted protein degradation mediated by IMiDs via CRBN emerged and developed rapidly for the advantages of overcoming drug resistance and targeting undruggable. To date, almost all CRBN ligands are derived from thalidomide and there are few structural differences between them. Hence, we employed an accurate, effective, and rational approach to screen novel and potential CRBN ligands. In this study, we have built a molecular library by scaffold hopping with thalidomide. ADMET screening, virtual screening, and visual inspection screening were performed step-by-step to screen the molecular library and five molecules were hit. Furthermore, docking analysis and a period of 150 ns molecular dynamic (MD) simulation were performed to validate the accuracy of our screen. The docking results showed that molecular A (-10.42 kcal/mol), molecular B (-9.73 kcal/mol), molecular C (-9.25 kcal/mol), molecular D (-9.09 kcal/mol), and molecular E (-10.16 kcal/mol) have lower binding energy than thalidomide (-5.42 kcal/mol), lenalidomide (-5.74 kcal/mol), and pomalidomide (-5.51 kcal/mol). In the MD simulation, all the five screened molecules form key interactions with the active site amino acid residues (Trp380, Trp386, and Trp400) as well as the three marketed IMiDs. Besides, we found and explained that Pro352 was positive for ligand binding to CRBN and Glu377 in reverse, which has not been reported before. We believe that our findings and those five molecules can serve as further optimization of CRBN ligands and development of proteolysis targeting chimeras.

Engineering spatial-organized cardiac organoids for developmental toxicity testing.

Emerging technologies in stem cell engineering have produced sophisticated organoid platforms by controlling stem cell fate via biomaterial instructive cues. By micropatterning and differentiating human induced pluripotent stem cells (hiPSCs), we have engineered spatially organized cardiac organoids with contracting cardiomyocytes in the center surrounded by stromal cells distributed along the pattern perimeter. We investigated how geometric confinement directed the structural morphology and contractile functions of the cardiac organoids and tailored the pattern geometry to optimize organoid production. Using modern data-mining techniques, we found that pattern sizes significantly affected contraction functions, particularly in the parameters related to contraction duration and diastolic functions. We applied cardiac organoids generated from 600 µm diameter circles as a developmental toxicity screening assay and quantified the embryotoxic potential of nine pharmaceutical compounds. These cardiac organoids have potential use as an in vitro platform for studying organoid structure-function relationships, developmental processes, and drug-induced cardiac developmental toxicity.

Progressive Myofibril Reorganization of Human Cardiomyocytes on a Dynamic Nanotopographic Substrate.

Cardiomyocyte (CM) alignment with striated myofibril organization is developed during early cardiac organogenesis. Previous work has successfully achieved in vitro CM alignment using a variety of biomaterial scaffolds and substrates with static topographic features. However, the cellular processes that occur during the response of CMs to dynamic surface topographic changes, which may provide a model of in vivo developmental progress of CM alignment within embryonic myocardium, remains poorly understood. To gain insights into these cellular processes involved in the response of CMs to dynamic topographic changes, we developed a dynamic topographic substrate that employs a shape memory polymer coated with polyelectrolyte multilayers to produce a flat-to-wrinkle surface transition when triggered by a change in incubation temperature. Using this system, we investigated cellular morphological alignment and intracellular myofibril reorganization in response to the dynamic wrinkle formation. Hence, we identified the progressive cellular processes of human-induced pluripotent stem cell-CMs in a time-dependent manner, which could provide a foundation for a mechanistic model of cardiac myofibril reorganization in response to extracellular microenvironment changes.

Effect of freezing on recombinant hepatitis E vaccine.

Studies have revealed that vaccines are more often exposed to sub-zero temperatures during cold chain transportation than what was previously known. Such exposure might be detrimental to the potency of temperature-sensitive vaccines. The aim of this study was to evaluate the impact of exposure to freezing on the physicochemical properties and biological activities of recombinant hepatitis E (rHE) vaccine. Changes in rHE vaccine due to freezing temperatures were analyzed with regard to sedimentation rate, antigenicity, and antibody affinity and potency. The freezing temperature of rHE was measured, then rHE vaccine was exposed to freezing temperatures below -10°C.Significant increase of sedimentation rate was noted, according to shake test and massed precipitates. In addition, the binding affinity of rHE vaccine to six specific monoclonal antibodies was significantly reduced and the in vivo potency for eliciting a protective IgG response was also partially lost, especially for anti-HEV neutralizing antibodies. Altogether, our work indicates that exposure of rHE vaccine to a temperature below -10°C results in the loss of structural integrity and biological potency of rHE vaccine.