Yongli Yu

This study explores the role of succinate as a vaccine adjuvant, revealing its capacity to enhance neutrophil recruitment at immunization sites, which boosts B cell activation through the succinate receptor 1-interferon regulatory factor 5-B cell-activating factor (SUCNR1-IRF5-BAFF) signaling pathway. Results demonstrate succinate’s potential to amplify vaccine-induced antibody responses, highlighting its significance in immunological enhancement and offering new insights into the adjuvant mechanisms of action, particularly in neutrophil-mediated immune responses.

Neutrophils exert either pro- or anti-tumor activities. However, few studies have focused on neutrophils at the tumor initiation stage. In this study, we unexpectedly found a subcutaneous nodule in the groin areas of mice inoculated with tumor cells. The nodule was developed 24 h after the inoculation, filled with tumor cells and massively recruited neutrophils, being designated as tumor nodules. 22% of the neutrophils in tumor nodules are surface TLR9 (sTLR9) expressing neutrophils (sTLR9⁺ neutrophils). With tumor progression, sTLR9⁺ neutrophils were sustainably increased in tumor nodules/tumor tissues, reaching to 90.8% on day 13 after inoculation, with increased expression of IL-10 and decreased or no expression of TNFα. In vivo administration of CpG 5805 significantly reduced sTLR9 expression of the sTLR9⁺ neutrophils. The reduction of sTLR9 on neutrophils in tumor nodules contributed to the induction of an anti-tumor microenvironment conductive to the inhibition of tumor growth. Overall, the study provides insights for understanding the role of sTLR9⁺ neutrophils in the tumor development, especially in the early stage. Graphical abstract

Introduction: Transforming growth factor β2 (TGF-β2), also known as glioma-derived T-cell suppressor factor, is associated with the impairment of tumor immune surveillance. Therefore, blocking TGF-β2 signaling probably be a feasible strategy to develop a novel type of adjuvant for glioma vaccines to enhance antitumor immunity.Methods: A TGF-β2 inhibitory oligodeoxynucleotide, TIO3, was designed with sequences complementary to the 3' untranslated region of TGF-β2 mRNA. The expression of TGF-β2 and MHC-I was detected by qPCR, western and flow cytometry in vitro. All the percentage and activation of immune cells were detected by flow cytometry. Subsequently, TIO3 was formulated with Glioma cell lysate (TCL) and investigated for its antitumor effects in GL261 murine glioma prophylactic and therapeutic models.Results: TIO3 could efficiently downregulate the expression of TGF-β2 while increase the MHC-I's expression in GL261 and U251 glioma cells in vitro. Meanwhile, TIO3 was detected in mice CD4+ T, CD8+ T, B and Ly6G+ cells from lymph nodes after 24 hours incubation. Moreover, TCL+TIO3 vaccination significantly prolonged the survival of primary glioma-bearing mice and protected these mice from glioma re-challenge in vivo. Mechanistically, TCL+TIO3 formulation strongly evoke the antitumor immune responses. 1) TCL+TIO3 significantly increased the composition of CD4+ and CD8+ T cells from draining lymph nodes while promoted their IFN-γ production and reduced the expression of TGF-β2 and PD1. 2) TCL+TIO3 activated the NK cells with the elevation of CD69 or NKG2D expression and PD1 reduction. 3) TCL+TIO3 increased the glioma-specific lysis CTLs from spleen. 4) TCL+TIO3 downregulated PD-L1 expression in glioma tissues and in Ly6G+ cells among glioma-infiltrating immune cells.Conclusion: TIO3 is a promising adjuvant for enhancing TCL-based vaccines to produce a more vigorous and long-lasting antitumor response by interfering with TGF-β2 expression.

Tumor immunotherapies have shown promising antitumor effects, especially immune checkpoint inhibitors (ICIs). However, only 12.46% of the patients benefit from the ICIs, the rest of them shows limited effects on ICIs or even accelerates the tumor progression due to the lack of the immune cell infiltration and activation in the tumor microenvironment (TME). In this study, we administrated a combination of Toll-like receptor 9 (TLR9) agonist CpG ODN and Transforming growth factor-β2 (TGF-β2) antisense oligodeoxynucleotide TIO3 to mice intraperitoneally once every other day for a total of four injections, and the first injection was 24 h after LLC cell inoculation. We found that the combination induced the formation of TME toward the enrichment and activation of CD8⁺ T cells and NK cells, accompanied with a marked decrease of TGF-β2. The combined therapy also effectively inhibited the tumor growth and prolonged the survival of the mice, even protected the tumor-free mice from the tumor re-challenge. Both of CpG ODN and TIO3 are indispensable, because replacing CpG ODN with TLR9 inhibitor CCT ODN showed no antitumor effect, CpG ODN or TIO3 alone did not lead to ideal antitumor results. This effect was possibly initiated by the activation of dendritic cells at the tumor site. This systemic antitumor immunotherapy with a combination of the two oligonucleotides (an immune stimulant and an immunosuppressive cytokine inhibitor) before the tumor formation may provide a novel strategy for clinical prevention of the postoperative tumor recurrence. Graphical abstract

Objectives To develop a method for in vitro assembly of recombinant proteins expressed in E. coli into chimeric virus-like particles (cVLPs). Results A fusion protein (Bepi-Cap-A) between capsid protein (Cap) of PCV2b and B cell epitope (Bepi) of IBDV was expressed in E. Coli, and purified. For assembling them into cVLPs (Bepi-Cap-VLP), the Bepi-Cap-A was suspended in buffer C [0.03% (“%” stands for “v/v” unless otherwise indicated) polyethylene glycol, 0.4 M Tris, 10 mM β-mercaptoethanol, 5% glycerol, 0.02% (w/v) gellan gum, 0.1 M glycine, 0.03% Tween 80, 500 mM NaCl], and incubated. After centrifugation, the pellet was resuspended in buffer D [50 mM Na₂HPO₄, 50 mM NaH₂PO₄, 0.01% (w/v) gellan gum, 0.05 mM EDTA, 500 mM NaCl, 0.03% Tween 80, pH 6.5], and then dialyzed against dialysis buffer (50 mM Na₂HPO₄, 50 mM NaH₂PO₄, 500 mM NaCl, 0.03% Tween 80, pH 6.5). The procedure resulted in typical and immunogenic Bepi-Cap-VLP. Conclusions The data provide a method which is feasible for in vitro assembly of recombinant proteins into chimeric virus-like particles.

Background: Finding conservative T cell epitopes in the proteome of numerous variants of SARS-COV-2 is required to develop T cell activating SARS-COV-2 capable of inducing T cell responses against SARS-COV-2 variants. Methods: A computational workflow was performed to find HLA restricted CD8 ⁺ and CD4 ⁺ T cell epitopes among conserved amino acid sequences across the proteome of 474727 SARS-CoV-2 strains. Results: A batch of covserved regions in the amino acid sequences were found in the proteome of the SARS-COV-2 strains. 2852 and 847 peptides were predicted to have high binding affinity to distint HLA class I and class II molecules. Among them, 1456 and 484 peptides are antigenic. 392 and 111 of the antigenic peptides were found in the conseved amino acid sequences. Among the antigenic-conserved peptides, 6 CD8 ⁺ T cell epitopes and 7 CD4 ⁺ T cell epitopes were identifed. The T cell epitopes could be presented to T cells by high-affinity HLA molecules which are encoded by the HLA alleles with high population coverage. Conclusions: The T cell epitopes are conservative, antigenic and HLA presentable, and could be constructed into SARS-COV-2 vaccines for inducing protective T cell immunity against SARS-COV-2 and their variants.