Oncolytic α-herpesvirus and myeloid-tropic cytomegalovirus cooperatively enhance systemic antitumor responses.

Oncolytic virotherapy aims to activate host antitumor immunity. In responsive tumors, intratumorally injected herpes simplex viruses (HSVs) have been shown to lyse tumor cells, resulting in local inflammation, enhanced tumor antigen presentation, and boosting of antitumor cytotoxic lymphocytes. In contrast to HSV, cytomegalovirus (CMV) is nonlytic and reprograms infected myeloid cells, limiting their antigen-presenting functions and protecting them from recognition by natural killer (NK) cells. Here, we show that when co-injected into mouse tumors with an oncolytic HSV, mouse CMV (mCMV) preferentially targeted tumor-associated myeloid cells, promoted the local release of proinflammatory cytokines, and enhanced systemic antitumor immune responses, leading to superior control of both injected and distant contralateral tumors. Deletion of mCMV genes m06, which degrades major histocompatibility complex class I (MHC class I), or m144, a viral MHC class I homolog that inhibits NK activation, was shown to diminish the antitumor activity of the HSV/mCMV combination. However, an mCMV recombinant lacking the m04 gene, which escorts MHC class I to the cell surface, showed superior HSV adjuvanticity. CMV is a potentially promising agent with which to reshape and enhance antitumor immune responses following oncolytic HSV therapy.

MeV-Stealth: A CD46-specific oncolytic measles virus resistant to neutralization by measles-immune human serum.

The frequent overexpression of CD46 in malignant tumors has provided a basis to use vaccine-lineage measles virus (MeV) as an oncolytic virotherapy platform. However, widespread measles seropositivity limits the systemic deployment of oncolytic MeV for the treatment of metastatic neoplasia. Here, we report the development of MeV-Stealth, a modified vaccine MeV strain that exhibits oncolytic properties and escapes antimeasles antibodies in vivo. We engineered this virus using homologous envelope glycoproteins from the closely-related but serologically non-cross reactive canine distemper virus (CDV). By fusing a high-affinity CD46 specific single-chain antibody fragment (scFv) to the CDV-Hemagglutinin (H), ablating its tropism for human nectin-4 and modifying the CDV-Fusion (F) signal peptide we achieved efficient retargeting to CD46. A receptor binding affinity of ~20 nM was required to trigger CD46-dependent intercellular fusion at levels comparable to the original MeV H/F complex and to achieve similar antitumor efficacy in myeloma and ovarian tumor-bearing mice models. In mice passively immunized with measles-immune serum, treatment of ovarian tumors with MeV-Stealth significantly increased overall survival compared with treatment with vaccine-lineage MeV. Our results show that MeV-Stealth effectively targets and lyses CD46-expressing cancer cells in mouse models of ovarian cancer and myeloma, and evades inhibition by human measles-immune serum. MeV-Stealth could therefore represent a strong alternative to current oncolytic MeV strains for treatment of measles-immune cancer patients.

Evidence for Angiotensin II as a Naturally Existing Suppressor for the Guanylyl Cyclase A Receptor and Cyclic GMP Generation.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Glucose Starvation Causes ptauS409 Increase in N2a Cells Through ATF3/PKAcα Signaling Pathway.

In this work, we report that glucose starvation (GS) causes ptauS409 increase, which may participate in GS-induced neurites retraction in neuro-2a (N2a) cells. Upon GS treatment, PKAcα was stimulated at mRNA and protein levels. Luciferase reporter gene assays indicated that GS regulated PKAcα expression through a core promoter (-345 to -95 bp upstream the transcription starting site) consisting of a cis-acting element of Activating Transcription Factor 3 (ATF3). Knockdown and over-expression experiments demonstrate that ATF3 transcriptionally regulated PKAcα expression. Moreover, GS stimulated ATF3 expression in a time-dependent manner. These findings reveal that glucose starvation induces ptauS409 increase in N2a cells through an ATF3- PKAcα axis, which shed some light on the relationship between brain glucose metabolism and neurodegenerative diseases.

Targeting glycogen synthase kinase 3 for therapeutic benefit in lymphoma.

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and ß are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3ß compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3ß genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3ß binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3ß is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3ß correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

O-GlcNAcylation of Thr12/Ser56 in short-form O-GlcNAc transferase (sOGT) regulates its substrate selectivity.

O-GlcNAcylation is a ubiquitous protein glycosylation playing different roles on variant proteins. O-GlcNAc transferase (OGT) is the unique enzyme responsible for the sugar addition to nucleocytoplasmic proteins. Recently, multiple O-GlcNAc sites have been observed on short-form OGT (sOGT) and nucleocytoplasmic OGT (ncOGT), both of which locate in the nucleus and cytoplasm in cell. Moreover, O-GlcNAcylation of Ser389 in ncOGT (1036 amino acids) affects its nuclear translocation in HeLa cells. To date, the major O-GlcNAcylation sites and their roles in sOGT remain unknown. Here, we performed LC-MS/MS and mutational analyses to seek the major O-GlcNAcylation site on sOGT. We identified six O-GlcNAc sites in the tetratricopeptide repeat domain in sOGT, with Thr12 and Ser56 being two "key" sites. Thr12 is a dominant O-GlcNAcylation site, whereas the modification of Ser56 plays a role in regulating sOGT O-GlcNAcylation, partly through Thr12In vitro activity and pulldown assays demonstrated that O-GlcNAcylation does not affect sOGT activity but does affect sOGT-interacting proteins. In HEK293T cells, S56A bound to and hence glycosylated more proteins in contrast to T12A and WT sOGT. By proteomic and bioinformatics analyses, we found that T12A and S56A differed in substrate proteins (e.g. HNRNPU and PDCD6IP), which eventually affected cell cycle progression and/or cell proliferation. These findings demonstrate that O-GlcNAcylation modulates sOGT substrate selectivity and affects its role in the cell. The data also highlight the regulatory role of O-GlcNAcylation at Thr12 and Ser56.

Rapid and sensitive MALDI MS analysis of oligosaccharides by using 2-hydrazinopyrimidine as a derivative reagent and co-matrix.

Sensitive analysis of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is significantly hampered by the low ionization efficiency of oligosaccharides. Derivatization affords a feasible way to enhance the MALDI intensities of oligosaccharides by introducing an easily ionized and/or hydrophobic tag to their reducing ends. However, tagging and subsequent desalting processes are quite time-consuming. Herein, we develop a rapid and sensitive approach for oligosaccharide derivatization by using 2-hydrazinopyrimidine (2-HPM). As a result of the presence of an electron-withdrawing N-heterocycle, 2-HPM can quantitatively derivatize oligosaccharides within 15 min and selectively facilitate their ionization. Additionally, 2-HPM acts as co-matrix to enhance the MALDI signal of oligosaccharides, and therefore the tedious enrichment and purification processes prior to MALDI analysis are avoided. This approach is applied to the analysis of various oligosaccharides released from glycopeptides, glycoprotein, and biological samples. After derivatization, a significant increase of MALDI intensities (greater than 10-fold) was observed for all the tested neutral and sialylated oligosaccharides. Moreover, the enhanced fragmentation of MS/MS brings much convenience to the structural elucidation of oligosaccharides. Graphical abstract Improved MALDI MS analysis of oligosaccharides by using 2-hydrazinopyrimidine as a derivative tag and co-matrix.

Robust Oncolytic Virotherapy Induces Tumor Lysis Syndrome and Associated Toxicities in the MPC-11 Plasmacytoma Model.

Tumor-selective oncolytic vesicular stomatitis viruses (VSVs) are being evaluated in clinical trials. Here, we report that the MPC-11 murine plasmacytoma model is so extraordinarily susceptible to oncolytic VSVs that a low dose of virus leads to extensive intratumoral viral replication, sustained viremia, intravascular coagulation, and a rapidly fatal tumor lysis syndrome (TLS). Rapid softening, shrinkage and hemorrhagic necrosis of flank tumors was noted within 1-2 days after virus administration, leading to hyperkalemia, hyperphosphatemia, hypocalcemia, hyperuricemia, increase in plasma cell free DNA, lymphopenia, consumptive coagulopathy, increase in fibrinogen degradation products, decreased liver function tests, dehydration, weight loss, and euthanasia or death after 5-8 days. Secondary viremia was observed but viral replication in normal host tissues was not detected. Toxicity could be mitigated by using VSVs with slowed replication kinetics, and was less marked in animals with smaller flank tumors. The MPC-11 tumor represents an interesting model to further study the complex interplay of robust intratumoral viral replication, tumor lysis, and associated toxicities in cases where tumors are highly responsive to oncolytic virotherapy.

Structural and biochemical insights into nucleotide-rhamnose synthase/epimerase-reductase from Arabidopsis thaliana.

L-Rhamnose (Rha) is synthesized via a similar enzymatic pathway in bacteria, plants and fungi. In plants, nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER) catalyzes the final step in the conversion of dTDP/UDP-α-D-Glc to dTDP/UDP-ß-L-Rha in an NAD(P)H dependent manner. Currently, only biochemical evidence for the function of NRS/ER has been described. In this study, a crystal structure for Arabidopsis thaliana NRS/ER was determined, which is the first report of a eukaryotic rhamnose synthase with both epimerase and reductase activities. NRS/ER functions as a metal ion independent homodimer that forms through hydrophobic interactions via a four-helix bundle. Each monomer exhibits α/ß folding that can be divided into two regions, nucleotide cofactor binding domain and sugar substrate binding domain. The affinities of ligands with NRS/ER were measured using isothermal titration calorimetry, which showed that NRS/ER has a preference for dTDP over UDP, while the cofactor binding site has a similar affinity for NADH and NADPH. Structural analysis coupled to site-directed mutagenesis suggested C115 and K183 as the acid/base pair responsible for epimerization, while T113, Y144 and K148 are the conserved residues in reduction. These findings shed light on the molecular mechanism of NRS/ER and were helpful to explore other eukaryotic enzymes involved in L-Rha synthesis.

O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway.

O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation.

Site-Directed Glycosylation of Peptide/Protein with Homogeneous O-Linked Eukaryotic N-Glycans.

Here we report a facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. A pharmaceutical peptide, glucagon-like peptide-1 (GLP-1), and a model protein, bovine α-Crystallin, were successfully glycosylated using such an approach. It was shown that the GLP-1 with O-linked N-glycan maintained an unchanged secondary structure after glycosylation, suggesting the potential application of this approach for peptide/protein drug production. In summary, the coupled approach provides a general strategy to produce homogeneous glycopeptide/glycoprotein bearing eukaryotic N-glycans.

Proteomic analysis of O-GlcNAcylated proteins in invasive ductal breast carcinomas with and without lymph node metastasis.

The potential role of protein O-GlcNAcylation in cancer has been studied extensively, and the spread of cancer cells to regional lymph nodes is the first step in the dissemination of breast cancer. However, the correlation between O-GlcNAcylation and lymphatic metastasis in breast cancer remains elusive. In this study, we demonstrated that the overall O-GlcNAcylation as well as O-GlcNAc transferase (OGT) tends to decrease in response to the augmentation of lymph node metastasis (LNM) in invasive ductal breast carcinomas (IDCs). Although accumulating evidence indicates that individual O-GlcNAcylation may be important in the pathogenesis of breast cancer, O-GlcNAcylated proteins in IDCs are still largely unexplored. Herein, O-GlcNAcylated proteins of IDCs were chemo-enzymatically enriched and identified via liquid chromatography combined with tandem mass spectrometry. In total, 155 O-GlcNAcylated proteins were determined, of which 41 were only observed in LNM tissues, while 40 were unique in non-LNM samples. Gene ontology analysis showed that O-GlcNAc is primarily a nucleocytoplasmic post-translational modification, and most enriched functional terms were related to cancer development in both metastatic and non-metastatic IDCs. Moreover, several O-GlcNAcylated proteins involved in glycolysis and its accessory pathway were identified from LNM and non-LNM groups, respectively. These results indicate that the O-GlcNAcylation statuses of individual proteins were independent of the overall O-GlcNAcylation levels of metastatic and non-metastatic IDCs. Aberrant O-GlcNAc modification of these proteins might be associated with LNM progression.

A peptide panel investigation reveals the acceptor specificity of O-GlcNAc transferase.

O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is widely distributed on nucleocytoplasmic proteins and participates in various physiological processes. But O-GlcNAc status on numerous proteins remains unknown. To better understand this modification, computational analysis combined with experimental study was performed in this work. Structural analysis of many O-GlcNAcylation sites indicated that the modification occurred predominantly in a random coil region. Frequency analysis on many O-GlcNAcylated peptides revealed a signature sequence, PPVS/TSATT, around the modification site (underlined, position 0). Based on the sequence, a peptide panel was designed to investigate key positions affecting O-GlcNAcylation of peptides and their amino acid preference. It was indicated that 3 positions (-2, -1, and +2) had an important role for this modification, where the presence of uncharged amino acids with small side chains could confer high reactivity. The amino acid preference at key positions was further investigated on bovine crystalline α via site-directed mutagenesis. The preferred amino acids were Pro > Ala > Gly at position -2, Ala > Thr > Val > Lys > Pro at position -1, and Ala > Gly > Arg > Glu at position +2. Altogether, these findings suggested that a substrate (peptide or protein) with Pro, Ala at position -2, and/or Val, Ala, Thr, Ser at position -1, and/or Ala, Ser, Pro, Thr, Gly at position +2 would have more chances for O-GlcNAcylation. To test the rule, 2 O-GlcNAcylation sites on sOGT (S52 and T449) were predicted and confirmed by Western blot. The present work systematically investigated the sequence signature for O-GlcNAcylation. The result will contribute to predicting the O-GlcNAc status of a protein and further functional studies.

Oncolytic varicella-zoster virus engineered with ORF8 deletion and armed with drug-controllable interleukin-12.

BACKGROUND: The varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a potential association between VZV infection and cancer remission. METHODS: Here, we constructed oncolytic VZV candidates based on the vaccine strain vOka and the laboratory strain Ellen. These newly engineered viruses were subsequently assessed for their oncolytic properties in the human MeWo melanoma xenograft model and the mouse B16-F10-nectin1 melanoma syngeneic model. RESULTS: In the MeWo xenograft model, both vOka and Ellen exhibited potent antitumor efficacy. However, it was observed that introducing a hyperfusogenic mutation into glycoprotein B led to a reduction in VZV's effectiveness. Notably, the deletion of ORF8 (encodes viral deoxyuridine triphosphatase) attenuated the replication of VZV both in vitro and in vivo, but it did not compromise VZV's oncolytic potency. We further armed the VZV Ellen-ΔORF8 vector with a tet-off controlled mouse single-chain IL12 (scIL12) gene cassette. This augmented virus was validated for its oncolytic activity and triggered systemic antitumor immune responses in the immunocompetent B16-F10-nectin1 model. CONCLUSIONS: These findings highlight the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.

Surface-modified measles vaccines encoding oligomeric, prefusion-stabilized SARS-CoV-2 spike glycoproteins boost neutralizing antibody responses to Omicron and historical variants, independent of measles seropositivity.

Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

EVIDENCE FOR ANGIOTENSIN II AS A NATURALLY EXISTING SUPPRESSOR FOR THE NATRIURETIC PEPTIDE SYSTEM.

Background: Natriuretic peptide system (NPS) and renin angiotensin aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date support this notion. Objectives: This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro for translational insights. Methods: Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in rat model to determine influence of ANGII on ANP actions. Multiple engineered HEK293 cells and surface plasmon resonance (SPR) technology were leveraged for mechanistic exploration. Results: In humans, ANGII showed inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and interaction term between ANGII and natriuretic peptide increased predicting accuracy of base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed positive association between cGMP with ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at physiological dose attenuated blood pressure reduction and cGMP generation triggered by ANP infusion. In vitro, we showed that the suppression effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), which can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using SPR, we showed ANGII has low affinity for particulate guanylyl cyclase A (GC-A) receptor binding compared to ANP or BNP. Conclusions: Our study reveals ANGII as a natural suppressor for cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular disease.

A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses.

Introduction: Metastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM. Methods: We developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-ß) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNß-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNß-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective. Results: 12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses. Discussion: Our study found that VSV-IFNß -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNß-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen.

Synthesis of flavonol 3-O-glycoside by UGT78D1.

Glycosylation is an important method for the structural modification of various flavonols, resulting in the glycosides with increased solubility, stability and bioavailability compared with the corresponding aglycone. From the physiological point of view, glycosylation of plant flavonoids is of importance and interest. However, it is notoriously complicated that flavonols such as quercetin, kaempferol and myricetin, are glucosylated regioselectively at the specific position by chemical method. Compared to the chemical method, enzymatic synthesis present several advantages, such as mild reaction condition, high stereo or region selectivity, no protection/deprotection and high yield. UGT78D1 is a flavonol-specific glycosyltransferase, responsible for transferring rhamnose or glucose to the 3-OH position in vitro. In this study, the activity of UGT78D1 was tested against 28 flavonoids acceptors using UDP-glucose as donor nucleoside in vitro, and 5 acceptors, quercetin, myricetin, kaempferol, fisetin and isorhamnetin, were discovered to be glucosylated at 3-OH position. Herein, the small-scale 3-O-glucosylated quercetin, kaempferol and myricetin were synthesized by UGT78D1 and their chemical structures were confirmed by (1)H and (13)C nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS).

HERC5/IFI16/p53 signaling mediates breast cancer cell proliferation and migration.

AIMS: This study aims to find differentially expressed ubiquitination-related gene(s) and elucidates their biological significance in breast cancer. MAIN METHODS: Differentially expressed genes were profiled in MCF-7 and MDA-MB-231 cells by using PCR array method. Abnormal expression of HERC5 was studied in the cells and in breast cancer specimens via Quantitative Real-time PCR and western blot. Cell proliferation and cell migration abilities were evaluated by using cell counting kits, or through colony formation, wound healing and trans-well assays. HERC5 target proteins were investigated via proteomic, co-immunoprecipitation and western blot methods. Down-stream signaling pathways were investigated through gene expression/knockdown methods. KEY FINDINGS: Huge increase of HERC5 expression was found in MCF-7 and MDA-MB-231 cells, knockdown of which repressed the cell proliferation and migration. HERC5 interacted with IFI16, mediated IFI16 ISGylation at K274 and facilitated IFI16 proteasomal degradation. IFI16 acted as a tumor suppressor and to some extent mediated the HERC5 function in the breast cancer (BC) cells. HERC5 was negatively correlated with IFI16 protein, while IFI16 was positively correlated to p53 expression at mRNA and protein levels, which indicates a novel signaling pathway - HERC5/IFI16/p53. HERC5 expression was increased in glucose-starved BC cells and in human breast cancer tissues, accompanied with the decrease of IFI16 and P53. SIGNIFICANCE: Our work reveals the abnormal expression of HERC5 and its carcinogenic role in breast cancer cells, which is probably mediated by an HERC5/IFI16/p53 signaling pathway. This work also provides potential diagnostic/therapeutic biomarkers for breast cancer diagnosis and treatment.

Surface-modified measles vaccines encoding oligomeric, fusion-stabilized SARS-CoV-2 spike glycoproteins bypass measles seropositivity, boosting neutralizing antibody responses to omicron and historical variants.

Serum titers of SARS-CoV-2 neutralizing antibodies (nAb) correlate well with protection from symptomatic COVID-19, but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are life-long after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We therefore sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated potent induction of high titer nAb in measles-immune mice and confirmed the significant incremental contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin-display of the SARS-CoV-2 spike glycoprotein, and vaccine resurfacing. In animals primed and boosted with a MeV vaccine encoding the ancestral SARS-CoV-2 spike, high titer nAb responses against ancestral virus strains were only weakly cross-reactive with the omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the omicron BA.1 spike, antibody titers to both ancestral and omicron strains were robustly elevated and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that antigen engineering can enable the development of potent measles-based SARS-CoV-2 vaccine candidates.