Strategy to develop broadly effective multivalent COVID-19 vaccines against emerging variants based on Ad5/35 platform.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron strain has evolved into highly divergent variants with several sub-lineages. These newly emerging variants threaten the efficacy of available COVID-19 vaccines. To mitigate the occurrence of breakthrough infections and re-infections, and more importantly, to reduce the disease burden, it is essential to develop a strategy for producing updated multivalent vaccines that can provide broad neutralization against both currently circulating and emerging variants. We developed bivalent vaccine AdCLD-CoV19-1 BA.5/BA.2.75 and trivalent vaccines AdCLD-CoV19-1 XBB/BN.1/BQ.1.1 and AdCLD-CoV19-1 XBB.1.5/BN.1/BQ.1.1 using an Ad5/35 platform-based non-replicating recombinant adenoviral vector. We compared immune responses elicited by the monovalent and multivalent vaccines in mice and macaques. We found that the BA.5/BA.2.75 bivalent and the XBB/BN.1/BQ.1.1 and XBB.1.5/BN.1/BQ.1.1 trivalent vaccines exhibited improved cross-neutralization ability compared to their respective monovalent vaccines. These data suggest that the developed multivalent vaccines enhance immunity against circulating Omicron subvariants and effectively elicit neutralizing antibodies across a broad spectrum of SARS-CoV-2 variants.

Boosting with variant-matched adenovirus-based vaccines promotes neutralizing antibody responses against SARS-CoV-2 Omicron sublineages in mice.

Global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron subvariants, such as BA.4, BA.5 and XBB.1.5, has been leading the recent wave of coronavirus disease 2019 (COVID-19). Unique mutations in the spike proteins of these emerging Omicron subvariants caused immune evasion from the pre-existing protective immunity induced by vaccination or natural infection. Previously, we developed AdCLD-CoV19-1, a non-replicating recombinant adenoviral vector that encodes the receptor binding domain of the spike protein of the ancestral SARS-CoV-2 strain. Based on the same recombinant adenoviral vector platform, updated vaccines that cover unique mutations found in each Omicron subvariant, including BA.1, BA.2, BA.4.1 and BA.5, were constructed. Preclinical studies revealed that each updated vaccine as a booster shot following primary vaccination targeting the ancestral strain improved neutralizing antibody responses against the pseudovirus of its respective strain most effectively. Of note, boosting with a vaccine targeting the BA.1 or BA.2 Omicron subvariant was most effective in neutralization against the pseudovirus of the BA.2.75 strain, whereas BA.4.1/5-adapted booster shots were most effective in neutralization against the BQ.1, BQ1.1 and BF.7 strains. Therefore, it is imperative to develop a vaccination strategy that can cover the unique spike mutations of currently circulating Omicron subvariants in order to prevent the next wave of COVID-19.

High-spatial and colourimetric imaging of histone modifications in single senescent cells using plasmonic nanoprobes.

Histones are closely related to the state of chromatin, and epigenetic modification of their tail results in regulation in cells. Therefore, developing various analytical tools to map the changes in position and distribution of histone modifications is helpful in studying underlying mechanisms. Herein, we propose a high-spatial and colourimetric imaging method using plasmonic nanoparticles as probes to visualize heterochromatin histone markers in a single nucleus. We visualized the reorganization between repressive histone markers, H3K9me3 and H3K27me3, caused by oncogene-induced senescence based on the scattering colours and spectral shift of plasmonic nanoprobes to longer wavelengths using their distance-dependent coupling effect. The measured scattering profiles were correlated with the computation results simulating the scattering spectra according to the arrangements and distances among the plasmonic nanoprobes. The plasmonic nanoprobe-based high-spatial hyperspectral imaging provides an advanced way to study the dynamics of histone modifications for predicting the progression of diseases or senescence.

HIF-1α-Dependent Induction of Carboxypeptidase A4 and Carboxypeptidase E in Hypoxic Human Adipose-Derived Stem Cells.

Hypoxia induces the expression of several genes through the activation of a master transcription factor, hypoxia-inducible factor (HIF)-1α. This study shows that hypoxia strongly induced the expression of two carboxypeptidases (CP), CPA4 and CPE, in an HIF-1α-dependent manner. The hypoxic induction of CPA4 and CPE gene was accompanied by the recruitment of HIF-1α and upregulation in the active histone modification, H3K4me3, at their promoter regions. The hypoxic responsiveness of CPA4 and CPE genes was observed in human adipocytes, human adipose-derived stem cells, and human primary fibroblasts but not mouse primary adipocyte progenitor cells. CPA4 and CPE have been identified as secreted exopeptidases that degrade and process other secreted proteins and matrix proteins. This finding suggests that hypoxia changes the microenvironment of the obese hypoxic adipose tissue by inducing the expression of not only adipokines but also peptidases such as CPA4 and CPE.

Hypoxia regulates allele-specific histone modification of the imprinted H19 gene.

H19 is a maternally-expressed imprinted gene that encodes long non-coding RNA. Chromatin immunoprecipitation (ChIP)-sequencing analyses of human adipose-derived stem cells (hADSCs) showed that hypoxia induced trimethylation of 4th lysine residue of histone 3 (H3K4me3) in the H19 gene, among the 40 known human imprinted genes, to the greatest extent. We investigated whether hypoxia changed the DNA and histone methylation levels of the imprinted H19 gene in an allele-specific (AS) manner. Using AS primer sets for the human H19 gene, we conducted ChIP-quantitative polymerase chain reaction, which revealed that hypoxia increased the active histone marks, H3K4me3 and H3K9/14Ac, in one allele (named B allele) but not in the other allele (named A allele). In contrast, hypoxia did not change the H3K9me3 levels in either allele. Hypoxia-inducible factor 1 (HIF-1) directly bound to the H19 promoter only in the B allele. HIF-1α knock-down prevented the increase in the active histone modification and mRNA expression of the B allele under hypoxia, indicating that HIF-1α caused AS changes in the histone modification of the H19 gene. Long-term hypoxia did not change the AS DNA methylation throughout the cell cycle. Thus, hypoxia changed the histone modification of the active allele in an HIF-1α-dependent manner, without changing the imprinted status of the H19 gene.

The cancer driver genes IDH1/2, JARID1C/ KDM5C, and UTX/ KDM6A: crosstalk between histone demethylation and hypoxic reprogramming in cancer metabolism.

Recent studies on mutations in cancer genomes have distinguished driver mutations from passenger mutations, which occur as byproducts of cancer development. The cancer genome atlas (TCGA) project identified 299 genes and 24 pathways/biological processes that drive tumor progression (Cell 173: 371-385 e318, 2018). Of the 299 driver genes, 12 genes are involved in histones, histone methylation, and demethylation (Table 1). Among these 12 genes, those encoding the histone demethylases JARID1C/KDM5C and UTX/KDM6A were identified as cancer driver genes. Furthermore, gain-of-function mutations in genes encoding metabolic enzymes, such as isocitrate dehydrogenases (IDH)1/2, drive tumor progression by producing an oncometabolite, D-2-hydroxyglutarate (D-2HG), which is a competitive inhibitor of α-ketoglutarate, O2-dependent dioxygenases such as Jumonji domain-containing histone demethylases, and DNA demethylases. Studies on oncometabolites suggest that histone demethylases mediate metabolic changes in chromatin structure. We have reviewed the most recent findings regarding cancer-specific metabolic reprogramming and the tumor-suppressive roles of JARID1C/KDM5C and UTX/KDM6A. We have also discussed mutations in other isoforms such as the JARID1A, 1B, 1D of KDM5 subfamilies and the JMJD3/KDM6B of KDM6 subfamilies, which play opposing roles in tumor progression as oncogenes or tumor suppressors depending on the cancer cell type. Table 1 Cancer driver genes involved in epigenetics Pathways involved in epigenetics Driver genes Tumor suppressor/oncogene prediction (by 20/20+a) Approved name Activity Cancer typeb Other driver genes in this pathways Histone modification KDM6A tsg Lysine demethylase 6A, UTX H3K27me2/3 demethylase BLCA, HNSC, KIRP, LUSC, PAAD, PANCAN, PRAD PPP6C SETD2 tsg SET domain-containing 2 H3K36 methyl transferase KIRC, KIRP, LGG, LUAD, MESO, PANCAN Chromatin histone modifiers KDM5C tsg Lysine demethylase 5C, JARID1C H3K4me2/3 demethylase KIRC, PANCAN ARID5B, CREBBP, EP300, KANSL1, MEN1, NCOR1, NSD1, SIN3A, WHSC1, ZMYM3 KMT2A tsg Lysine methyltransferase 2A H3K4 methyl transferase PANCAN KMT2B tsg Lysine methyltransferase 2B H3K4 methyl transferase PANCAN, UCEC KMT2C tsg Lysine methyltransferase 2C H3K4 methyl transferase BLCA, BRCA, CESC, PANCAN, UCEC KMT2D tsg Lysine methyltransferase 2D H3K4 methyl transferase BLCA, CESC, DLBC, ESCA, HNSC, LUSC, PANCAN, PRAD Chromatin (other) H3F3A Possible oncogene H3 histone family member 3A, H3.3A PANCAN AJUBA, ASXL1, ASXL2, ATF7IP, BCOR, CHD3, CHD4, CHD8, CTCF, NIPBL, NPM1 H3F3C - H3 histone family member 3C, H3.5 PANCAN HIST1H1E Possible oncogene HIST1H1E, H1.4 DLBC Possible tsg HIST1H1E, H1.4 LIHC Metabolism IDH1 Oncogene Isocitrate dehydrogenase (NADP(+)) 1 NADP-dependent IDH, Cytosolic CHOL, GBM, LAML, LGG, LIHC, PANCAN, PRAD, SKCM - IDH2 Oncogene Isocitrate dehydrogenase (NADP(+)) 2 NADP-dependent IDH, Mitochondrial LAML, LGG, PANCAN Among the 299 driver genes mentioned by Bailey et al.47, only the epigenetics-related pathways have been sorted out a20/20+: Classifies genes as an oncogene, tumor suppressor gene, or as a nondriver gene using Random Forests, http://2020plus.readthedocs.org bBLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), CHOL (cholangiocarcinoma), DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), ESCA (esophageal carcinoma), GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma), LAML (acute myeloid leukemia), LGG (brain lower grade glioma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), MESO (mesothelioma), PAAD (pancreatic adenocarcinoma), PANCAN (Pan-cancer), PRAD (prostate adenocarcinoma), SKCM (skin cutaneous melanoma), THCA (thyroid carcinoma), UCEC (uterine corpus endometrial carcinoma).

Wnt3a disrupts GR-TEAD4-PPARγ2 positive circuits and cytoskeletal rearrangement in a ß-catenin-dependent manner during early adipogenesis.

Adipogenesis is a process which induces or represses many genes in a way to drive irreversible changes of cell phenotypes; lipid accumulation, round cell-shape, secreting many adipokines. As a master transcription factor (TF), PPARγ2 induces several target genes to orchestrate these adipogenic changes. Thus induction of Pparg2 gene is tightly regulated by many adipogenic and also anti-adipogenic factors. Four hours after the treatment of adipogenic hormones, more than fifteen TFs including glucocorticoid receptor (GR), C/EBPß and AP-1 cooperatively bind the promoter of Pparg2 gene covering 400 bps, termed "hotspot". In this study, we show that TEA domain family transcription factor (TEAD)4 reinforces occupancy of both GR and C/EBPß on the hotspot of Pparg2 during early adipogenesis. Our findings that TEAD4 requires GR for its expression and for the ability to bind its own promoter and the hotspot region of Pparg2 gene indicate that GR is a common component of two positive circuits, which regulates the expression of both Tead4 and Pparg2. Wnt3a disrupts these mutually related positive circuits by limiting the nuclear location of GR in a ß-catenin dependent manner. The antagonistic effects of ß-catenin extend to cytoskeletal remodeling during the early phase of adipogenesis. GR is necessary for the rearrangements of both cytoskeleton and chromatin of Pparg2, whereas Wnt3a inhibits both processes in a ß-catenin-dependent manner. Our results suggest that hotspot formation during early adipogenesis is related to cytoskeletal remodeling, which is regulated by the antagonistic action of GR and ß-catenin, and that Wnt3a reinforces ß-catenin function.

Hypoxic reprograming of H3K27me3 and H3K4me3 at the INK4A locus.

Activation of Raf reduces the repressive histone mark H3K27me3 at the INK4a locus by inducing the H3K27me3 demethylase JMJD3. During hypoxia, the catalyitc activity of JMJD3 is reduced due to the limited availability of O2 as a substrate. In our study, we found that hypoxia prevented Raf-induced JMJD3 from demethylating H3K27me3 at the INK4a locus. Nonetheless, hypoxia did not prevent Raf signaling from inducing INK4a mRNA. Interestingly, we found that hypoxia strongly enhanced the active histone mark H3K4me3 at the INK4a locus by inhibiting the H3K4me3 demethylases JARID1A and JARID1B. Therefore, this study demonstrates that the O2 concentration in the microenvironment differentially affects the repressive methylation on K27 and the activating methylation on K4 at the INK4a locus by inhibiting the H3K27me3 and H3K4me3 demethylases.

Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2) and severe hypoxia (0.1% O2). We found that chenodeoxy cholic acid (CDCA) reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR), a CDCA receptor and its target gene, Small heterodimer partner (SHP) are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.