Conjugation of TLR7 and TLR7/8 agonists onto weak protein antigen via versatile oxime ligation for enhanced vaccine efficacy.

Protein-based subunit vaccines are weakly immunogenic, and developing self-adjuvanting vaccines with adjuvant conjugated to antigen is a promising approach for generating optimal immune responses. Here, we report a novel adjuvant-protein conjugate vaccine based on versatile oxime ligation technique. Firstly, the adjuvant properties of a series of TLR7 and TLR7/8 small molecule agonists in self-adjuvanting vaccines were systematically compared by coupling them to proteins in consistent ratio via p-carboxybenzaldehyde (p-CBA) for the first time. All conjugate vaccines induced cytokine secretion in murine and human macrophages in vitro, and promoted specific antibody production in vivo. Notably, a conjugate containing imidazoquinoline TLR7/8 agonist (TLR7/8a1) showed the greatest enhancement in Th1/2 balanced antibody response. To minimize the interference with the protein antigenic integrity, we further developed a systematic glycoconjugation strategy to conjugate this TLR7/8a1 onto the glycan chains of SARS-CoV-2 S1 glycoprotein via oxime ligation, in which S1 containing different numbers of aldehyde groups were obtained by differential periodate oxidation. The resulting TLR7/8a1-S1 conjugate triggered a potent humoral and cellular immunity in vivo. Together these data demonstrate the promise of these TLR7 and TLR7/8 agonists as effective built-in adjuvants, and the versatile oxime ligation strategy might broaden potential applications in designing different conjugate vaccines.

Alum Adjuvant and Built-in TLR7 Agonist Synergistically Enhance Anti-MUC1 Immune Responses for Cancer Vaccine.

The tumor-associated antigen mucin 1 (MUC1) is an attractive target of antitumor vaccine, but its weak immunogenicity is a big challenge for the development of vaccine. In order to enhance immune responses against MUC1, herein, we conjugated small molecular toll-like receptor 7 agonist (TLR7a) to carrier protein BSA via MUC1 glycopeptide to form a three-component conjugate (BSA-MUC1-TLR7a). Furthermore, we combined the three-component conjugate with Alum adjuvant to explore their synergistic effects. The immunological studies indicated that Alum adjuvant and built-in TLR7a synergistically enhanced anti-MUC1 antibody responses and showed Th1-biased immune responses. Meanwhile, antibodies elicited by the vaccine candidate effectively recognized tumor cells and induced complement-dependent cytotoxicity. In addition, Alum adjuvant and built-in TLR7a synergistically enhanced MUC1 glycopeptide-specific memory CD8+ T-cell immune responses. More importantly, the vaccine with the binary adjuvant can significantly inhibit tumor growth and prolong the survival time of mice in the tumor challenge experiment. This novel vaccine construct provides an effective strategy to develop antitumor vaccines.

MUC1 Specific Immune Responses Enhanced by Coadministration of Liposomal DDA/MPLA and Lipoglycopeptide.

Mucin 1 (MUC1), a well-known tumor-associated antigen and attractive target for tumor immunotherapy, is overexpressed in most human epithelial adenomas with aberrant glycosylation. However, its low immunogenicity impedes the development of MUC1-targeted antitumor vaccines. In this study, we investigated three liposomal adjuvant systems containing toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) and auxiliary lipids of different charges: cationic lipid dimethyldioctadecylammonium (DDA), neutral lipid distearoylglycerophosphocholine (DSPC) or anionic lipid dioleoylphosphatidylglycerol (DOPG), respectively. ELISA assay evidenced that the positively charged DDA/MPLA liposomes are potent immune activators, which induced remarkable levels of anti-MUC1 antibodies and exhibited robust Th1-biased immune responses. Importantly, the antibodies induced by DDA/MPLA liposomes efficiently recognized and killed MUC1-positive tumor cells through complement-mediated cytotoxicity. In addition, antibody titers in mice immunized with P2-MUC1 vaccine were significantly higher than those from mice immunized with P1-MUC1 or MUC1 vaccine, which indicated that the lipid conjugated on MUC1 antigen also played important role for immunomodulation. This study suggested that the liposomal DDA/MPLA with lipid-MUC1 is a promising antitumor vaccine, which can be used for the immunotherapy of various epithelial carcinomas represented by breast cancer.

From design to clinic: Engineered peptide nanomaterials for cancer immunotherapy.

Immunotherapy has revolutionized the field of cancer therapy. Nanomaterials can further improve the efficacy and safety of immunotherapy because of their tunability and multifunctionality. Owing to their natural biocompatibility, diverse designs, and dynamic self-assembly, peptide-based nanomaterials hold great potential as immunotherapeutic agents for many malignant cancers, with good immune response and safety. Over the past several decades, peptides have been developed as tumor antigens, effective antigen delivery carriers, and self-assembling adjuvants for cancer immunotherapy. In this review, we give a brief introduction to the use of peptide-based nanomaterials for cancer immunotherapy as antigens, carriers, and adjuvants, and to their current clinical applications. Overall, this review can facilitate further understanding of peptide-based nanomaterials for cancer immunotherapy and may pave the way for designing safe and efficient methods for future vaccines or immunotherapies.

Autophagy inhibitors increase the susceptibility of KRAS-mutant human colorectal cancer cells to a combined treatment of 2-deoxy-D-glucose and lovastatin.

RAS-driven colorectal cancer relies on glucose metabolism to support uncontrolled growth. However, monotherapy with glycolysis inhibitors like 2-deoxy-D-glucose causes limited effectiveness. Recent studies suggest that anti-tumor effects of glycolysis inhibition could be improved by combination treatment with inhibitors of oxidative phosphorylation. In this study we investigated the effect of a combination of 2-deoxy-D-glucose with lovastatin (a known inhibitor of mevalonate pathway and oxidative phosphorylation) on growth of KRAS-mutant human colorectal cancer cell lines HCT116 and LoVo. A combination of lovastatin (>3.75 µM) and 2-deoxy-D-glucose (>1.25 mM) synergistically reduced cell viability, arrested cells in the G2/M phase, and induced apoptosis. The combined treatment also reduced cellular oxygen consumption and extracellular acidification rate, resulting in decreased production of ATP and lower steady-state ATP levels. Energy depletion markedly activated AMPK, inhibited mTOR and RAS signaling pathways, eventually inducing autophagy, the cellular pro-survival process under metabolic stress, whereas inhibition of autophagy by chloroquine (6.25 µM) enhanced the cytotoxic effect of the combination of lovastatin and 2-deoxy-D-glucose. These in vitro experiment results were reproduced in a nude mouse xenograft model of HCT116 cells. Our findings suggest that concurrently targeting glycolysis, oxidative phosphorylation, and autophagy may be a promising regimen for the management of RAS-driven colorectal cancers.

MicroRNA-144 represses gliomas progression and elevates susceptibility to Temozolomide by targeting CAV2 and FGF7.

Malignant gliomas are the most common tumor in central nervous system with poor prognosis. Due to the limitation of histological classification in earlier diagnosis and individualized medicine, it is necessary to combine the molecular signatures and the pathological characteristics of gliomas. Lots of microRNAs presented abnormal expression in gliomas and modulated gliomas development. Exploration the miRNAs profile is helpful for the diagnosis, therapy and prognosis of gliomas. It has been demonstrated that miR-144 plays important roles in solid tumors. However, the detail mechanisms remained unrevealed. In this study, we have demonstrated the level of miR-144 decreased in glioma tissues from patients, especially in gliomas with higher grades. MiR-144 was also validated have lower expression in glioma cell lines compared with cortical neuron cell by using qRT-PCR. The in vitro functional experiment indicated miR-144 improved gliomas progression through repressing proliferation, sensitizing to chemotherapeutics and inhibiting metastasis. We further identified fibroblast growth factor 7 (FGF7) and Caveolin 2 (CAV2) were target genes of miR-144 by luciferase reporter assay and western blotting. The mechanisms study suggested forced FGF7 expression elevated Akt activation and decreased reactive oxygen species (ROS) generation. The MTT and cell cycle assay indicated miR-144 suppressed glioma cells proliferation through modulating FGF mediated Akt signaling pathway. Meanwhile, miR-144 promoted Temozolomide (TMZ) induced apoptosis in glioma cells via increasing ROS production by using FACS. On the other hand, CAV2, as another target of miR-144, accelerated glioma cells migration and invasion via promoting glioma cells EMT progress. Retrieved expression of FGF7 or CAV2 rescued the proliferation and migration function mediated by miR-144. Furthermore, the in vivo experiments in PDX models displayed the anti-tumor function of miR-144, which could be retrieved by overexpression of FGF7 and CAV2. Taken together, these findings indicated miR-144 acted as a potential target against gliomas progression and uncovered a novel regulatory mechanism, which may provide a new therapeutic strategy and prognostic indicator for gliomas.

Multifunctional Protein Conjugates with Built-in Adjuvant (Adjuvant-Protein-Antigen) as Cancer Vaccines Boost Potent Immune Responses.

Many cancer vaccines are not successful in clinical trials, mainly due to the challenges associated with breaking immune tolerance. Herein, we report a new strategy using an adjuvant-protein-antigen (three-in-one protein conjugates with built-in adjuvant) as an anticancer vaccine, in which both the adjuvant (small-molecule TLR7 agonist) and tumor-associated antigen (mucin 1, MUC1) are covalently conjugated to the same carrier protein (BSA). It is shown that the protein conjugates with built-in adjuvant can increase adjuvant's stimulation, prevent adjuvant's systemic toxicities, facilitate the codelivery of adjuvants and antigens, and enhance humoral and cellular immune responses. The IgG antibody titers elicited by the self-adjuvanting three-in-one protein conjugates were significantly higher than those elicited by the vaccine mixed with TLR7 agonist (more than 15-fold) or other traditional adjuvants. Importantly, the potent immune responses against cancer cells suggest that this new vaccine construct is an effective strategy for the personalized antitumor immunotherapy.

Liposomal Antitumor Vaccines Targeting Mucin 1 Elicit a Lipid-Dependent Immunodominant Response.

The tumor-associated antigen mucin 1 (MUC1) has been pursued as an attractive target for cancer immunotherapy, but the poor immunogenicity of the endogenous antigen hinders the development of vaccines capable of inducing effective anti-MUC1 immunodominant responses. Herein, we prepared synthetic anti-MUC1 vaccines in which the hydrophilic MUC1 antigen was N-terminally conjugated to one or two palmitoyl lipid chains (to form amphiphilic Pam-MUC1 or Pam2 -MUC1). These amphiphilic lipid-tailed MUC1 antigens were self-assembled into liposomes containing the NKT cell agonist αGalCer as an adjuvant. The lipid-conjugated antigens reshaped the physical and morphological properties of liposomal vaccines. Promising results showed that the anti-MUC1 IgG antibody titers induced by the Pam2 -MUC1 vaccine were more than 30- and 190-fold higher than those induced by the Pam-MUC1 vaccine and the MUC1 vaccine without lipid tails, respectively. Similarly, vaccines with the TLR1/2 agonist Pam3 CSK4 as an adjuvant also induced conjugated lipid-dependent immunological responses. Moreover, vaccines with the αGalCer adjuvant induced significantly higher titers of IgG antibodies than vaccines with the Pam3 CSK4 adjuvant. Therefore, the non-covalent assembly of the amphiphilic lipo-MUC1 antigen and the NKT cell agonist αGalCer as a glycolipid adjuvant represent a synthetically simple but immunologically effective approach for the development of anti-MUC1 cancer vaccines.

Peptide-free Synthetic Nicotine Vaccine Candidates with α-Galactosylceramide as Adjuvant.

Peptides are generally needed as T-helper epitopes in nicotine vaccines to induce effective antibody responses, but the highly polymorphic property of major histocompatibility complex (MHC) molecules may limit opportunities of B cell to receive CD4+ T-cell help. Invariant natural killer T (iNKT) cells recognize lipid antigens presented by the nonpolymorphic CD1d molecule that is conserved in mammals to a great extent. iNKT cells also display some similar functions to conventional CD4+ T-helper cells, especially they license dendritic cells stimulate antibody isotype switching by B cells. Herein, α-galactosylceramide (αGalCer), a classical iNKT cell agonist, serves as an adjuvant in synthetic nicotine vaccine candidates absent of peptide or protein. Our study reveals that αGalCer displays better adjuvant activity than Pam3CSK4 (a commonly used lipopeptide TLR agonist). Remarkably, the covalent linker between the nicotine hapten and αGalCer is not critical. Self-assembly of the lipid-tailed nicotine and αGalCer into the liposome represents a structurally simple but immunologically effective way to develop nicotine vaccines. This is the first time to introduce the iNKT cell agonist as an adjuvant to an antidrug vaccine. This discovery may contribute to improving the efficacy of clinical candidate nicotine vaccines in the future.

Comparison between minimally invasive plate osteosynthesis and open plating for proximal humeral fractures: a meta-analysis.

AIM: We conducted this meta-analysis for comparing the efficacy and safety in proximal humeral fractures by treatment minimally invasive plate osteosynthesis and open plating. METHODS: The potential academic literature were identified from the Cochrane Library, Springer, PubMed, Embase and ScienceDirect. Pooled data were analyzed by RevMan 5.1. RESULTS: Seven studies marched with the inclusion criteria. Meta-analysis showed the significant differences in terms of blood loss, operative time, length of hospital stays and constant score between two groups. No significant differences were found in time to union, the union rate and complications. CONCLUSION: Minimally invasive plate osteosynthesis in proximal humeral fractures provided significantly shorter operative times, length of hospital stays, less blood loss and better clinical outcomes without increasing complications.

Visible-Light-Induced Specific Desulfurization of Cysteinyl Peptide and Glycopeptide in Aqueous Solution.

Visible-light-induced specific desulfurization of cysteinyl peptides has been explored. The photocatalytic desulfurization catalyzed by Ru(bpy)3(2+) can proceed efficiently at room temperature in aqueous solution or in binary mixtures of aqueous/organic solvent and be compatible with the presence of residues of amino acids, carbohydrates, and various sulfur-containing functional groups. This approach was successfully applied to synthesize linear and cyclic peptides through the ligation-desulfurization protocol.

Calcium-sensing receptor in the T lymphocyte enhanced the apoptosis and cytokine secretion in sepsis.

Calcium-sensing receptor (CaSR) is a member of the G protein-coupled receptor superfamily that existed in lymphocytes and promoted cytokine secretion. Lymphocytes are also involved in sepsis. However, the role of CaSR in lymphocytes in sepsis is unclear. In this study, we want to examine whether the CaSR in lymphocytes in sepsis is involved in the cytokine secretions and apoptosis and make clear the relationship between NF-κB and MAPK signal transduction pathways. We investigated the issues mentioned earlier using Western blotting, ELISA, and Flow Cytometry. The sepsis was remodeled by cecal ligation and puncture (CLP). We found that CaSR protein expression increased in the peripheral blood T lymphocytes in CLP rats. The calcimimetic R568 (NPS R568) promoted, whereas the calcilytic NPS 2143 attenuated, signaling pathways proteins P65 (subunit of NF-κB), ERK1/2, and JNK (one subgroup of MAPKs) phosphorylation. However, P-P38 and P-JAKs exhibit no significant changes. Furthermore, the production TNF-α and IL-4 was greater in CLP rats than in normal rats, and NPS R568 promoted secretion of these cytokines. Simultaneously, the apoptotic ratio of T cells in CLP increased, and NPS R 568 exacerbated the apoptosis degree. However, these effects could also be inhibited by U0126 or SP600125 (MAPKs pathway inhibitor) or Bay-11-7082 or (NF-κB pathway inhibitor). From these results, we can conclude that, in the sepsis, CaSR activation promoted T-cell apoptosis and the secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokines IL-4 probably through NF-κB and partial MAPK signal transduction pathways.

SHP2 promotes laryngeal cancer growth through the Ras/Raf/Mek/Erk pathway and serves as a prognostic indicator for laryngeal cancer.

The overall survival rate and prognosis of patients with laryngeal cancer are not optimistic despite advances in therapeutic techniques. Gene expression prognostic models enable the development of more appropriate treatment strategies. The human gene PTPN11 encoding a non-receptor protein tyrosine phosphatase, Src homology phosphotyrosine phosphatase 2 (SHP2), is a well-documented proto-oncogene in various malignancies. This study investigated the role of SHP2 expression and associated clinical manifestations in laryngeal cancer using a tissue microarray of 112 pairs of laryngeal cancer samples and corresponding adjacent normal mucosae. SHP2 expression increased in laryngeal cancer, and this result was associated with the poor survival rate of laryngeal cancer patients. Moreover, increased SHP2 expression remarkably promoted the growth of laryngeal cancer cells in vitro and tumorigenicity of laryngeal cancer cells in vivo. The Ras/Raf/Mek/Erk pathway was also found to be involved in the SHP2-induced growth of laryngeal cancer cells. Overall, our findings indicated that SHP2 plays an important role in laryngeal cancer tumorigenesis and that its expression is negatively correlated with the prognosis of patients. Thus, SHP2 may be a promising combinational therapeutic target for treatment of laryngeal cancer. The interference of SHP2 expression can serve as a novel strategy for laryngeal cancer treatment.