To prepare tubeimoside Ⅲ nanoemulsion (TBM Ⅲ-NE) and evaluate its adjuvant effect in vaccines.

TBMⅢ-NE was prepared using low-energy emulsification. Dynamic light scattering was used to characterize the particle size and polydispersity index of the obtained TBMⅢ-NE, and transmission electron microscopy (TEM) was employed to observe the morphology. CCK-8 assay was utilized to determine the cytotoxicity of TBMⅢ-NE on bone marrow-derived dendritic cells (BMDCs). The in vitro safety of TBMⅢ-NE was evaluated using a hemolysis assay. The ability of TBMⅢ-NE to promote the phagocytosis of antigens by DC2.4 cells was observed using confocal laser microscopy. After co-incubation of TBMⅢ-NE with BMDCs, the expression levels of CD40, CD86, MHC-Ⅰ, and CCR7 on the surface of BMDCs were detected using flow cytometry, and the levels of cytokines in the supernatant of BMDCs were measured using enzyme-linked immunosorbent assay (ELISA). After female BALB/c mice were immunized with the SARS-CoV-2 antigen RBD in combination with TBM Ⅲ-NE, ELISA was conducted to determine the serum levels of specific IgG, IgG2a, and IgG1 antibodies. The number of specific IFN-γ-secreting cells in mouse splenocytes was detected using enzyme-linked immunospot (ELISpot) assay.

The prepared blank nanoemulsion (BNE) and TBMⅢ-NE were in a particle size of 25.46 and 25.89 nm, and a polydispersity index of 0.214 and 0.125, respectively. TEM displayed that TBM Ⅲ-NE was in uniform sphere and well dispersed. When the TBMⅢ-NE adjuvant was diluted by 400-fold, the survival rate of BMDCs was approximately 86%. Compared with free TBM Ⅲ, the hemolytic toxicity of TBM Ⅲ-NE was significantly reduced (P < 0.01). TBMⅢ-NE promoted the phagocytosis of antigens by DC2.4 cells and significantly increased the expression of CCR7 on the surface of BMDCs (P < 0.05), indicating its potential to promote more dendritic cells to effectively migrate to lymph nodes. TBMⅢ-NE also promoted the expression of IL-6 and IL-1β in the supernatant of BMDCs (P < 0.05). When combined with RBD, TBMⅢ-NE significantly increased the levels of specific IgG, IgG2a, and IgG1 antibodies in mouse serum (P < 0.01) and promoted the secretion of specific IFN-γ in splenocytes (P < 0.01), indicating that TBM Ⅲ-NE could enhance specific cellular immune responses.

A stable and highly effective TBM Ⅲ-NE that can induce humoral and cellular immune responses is successfully prepared.

To investigate the interaction between a novel antimicrobial compound, HL-J6, and penicillin-binding protein 1 (PBP1) of Staphylococcus aureus.

With MRSA252 genomic DNA as the template and PBP1F and PBP1R as primers, the expression plasmid pET30a-pbp1-39-608 was constructed by amplifying the target gene fragment followed by cloning into the Nde Ⅰ/Xho Ⅰ restriction sites of the pET30a vector. Then the obtained plasmids were transformed into Escherichia coli for the expression of PBP1-39-608 protein, and the product was purified by affinity chromatography. The inhibitory effect of HL-J6 on the transpeptidase activity of PBP1-39-608 was measured using peptidoglycan side chain backbone peptide, with thiol ester analog S2d as the substrate. The affinity between HL-J6 and PBP1-39-608 was detected using microscale thermophoresis (MST), and the binding interaction was confirmed by cellular thermal shift assay (CETSA). Molecular docking and dynamics simulation were performed using AutoDock Vina and Desmond software, respectively, to elucidate the binding mode of HL-J6 with the PBP1-39-608 protein and the key amino acid residues involved.

The recombinant plasmid pET30a-pbp1-39-608 was successfully constructed, and PBP1-39-608 protein was produced after induction and purified, yielding a protein with an approximate molecular mass of 65×10³. HL-J6 inhibited the transpeptidase activity of PBP1-39-608 in a time-dependent manner (P<0.001). The dissociation constant Kd of the binding between HL-J6 and PBP1-39-608 was 64.92 μ mol/L. Molecular docking results showed that HL-J6 bound to the active pocket of PBP1-39-608 by interacting with key residues such as ILE-348, ASN-370, THR-516 and PHE-423, with a binding score of-8.38 kcal/mol (<-5.00 kcal/mol). Dynamics simulation results indicated that the complex became stable after 50 ns.

HL-J6 effectively inhibits the transpeptidase activity of Staphylococcus aureus PBP1, and shows stable interaction with the protein.

High-altitude chronic hypoxia can alter the immune status, yet the immune regulatory characteristics of various vaccine adjuvants in this environment remain to be fully elucidated. Accordingly, emphasis is placed on evaluating the regulatory effects of chronic hypoxia on specific humoral, cellular, and memory T cell responses elicited by different adjuvants.

Using ovalbumin (OVA) as the antigen, Th2-biased adjuvant aluminum hydroxide (Alum), Th1-biased adjuvant CpG, and Th1/Th2-balanced adjuvant QML were combined for intramuscular immunization in mice on days 0 and 14. Th1-biased adjuvant CpG, or Th1/Th2-balanced adjuvant QML for intramuscular immunization on day 0 and 14(n=16). A total of 160 SPF female C57 BL/6 mice (6 to 8 weeks old, weighing 16 to 18 g) were randomly divided into 5 groups (n=32): PBS group (sterile PBS), OVA group (5 μg OVA), OVA+Alum group (5 μg OVA and 100 μg Alum), OVA+CpG group (5 μg OVA and 20 μg CpG), and OVA+QML group (5 μg OVA and 50 μL QML). Each group was further divided into normoxia control (n=16, housed in 21% O₂ environment) and chronic hypoxia (n=16, simulated altitude 5800 m) subgroups. On days 14 and 60 after the last immunization, the serum levels of specific IgG, IgG1, and IgG2c antibodies were measured by ELISA; enzyme-linked immunospot assay (ELISpot) was used to detect the numbers of cells secreting IFN-γ, IL-4, and IL-17A in the splenocytes. On day 14 after the last immunization, flow cytometry was performed to determine the proportions of CD4⁺IFN-γ⁺, CD4⁺IL-4⁺, CD4⁺ IL-17A⁺, CD8⁺IFN-γ⁺, and CD8⁺IL-2⁺ cell subsets in the spleen.

Chronic hypoxia significantly inhibited the ability of OVA alone to induce IgG antibodies (P < 0.01), and all 3 adjuvants reversed this effect. QML exerted a stable and superior enhancing effect on serum-specific IgG, IgG1, and IgG2c levels compared to CpG (P < 0.05). Notably, CpG induced significantly higher levels of total IgG (P < 0.01) and IgG1(P < 0.05) in the chronic hypoxia group than in the corresponding normoxia group at days 60 after the final immunization. For cellular immunity, QML stably increased the proportions of CD4⁺IFN-γ⁺ and CD4⁺IL-17A⁺ cells, as well as the secretion levels of IFN-γ and IL-4, under both normoxic and hypoxic conditions. In contrast, the T cell regulatory effects of CpG were hypoxia-dependent, with significant enhancement of IFN-γ-secreting cell numbers in the hypoxia group at both short-term (14 d, P < 0.01) and longterm (60 d, P < 0.05) time points. Analysis of memory T cells showed that all 3 adjuvants maintained CD4⁺ Tcm levels under hypoxia. Additionally, CpG increased the proportions of CD8⁺ Tcm (P < 0.05) and CD8⁺ Tem (P < 0.01) in the hypoxic group, while Alum only specifically elevated CD4⁺ Tem levels under hypoxic conditions (P < 0.05).

QML stably induces comprehensive immune responses under both normoxic and hypoxic conditions, while CpG exhibits advantages in Th1-type response enhancement and CD8⁺ memory T cell expansion under chronic hypoxia, providing experimental evidence for adjuvant selection in chronic hypoxic environments such as high-altitude regions.

Helicobacter pylori (Hp) infection is prevalent worldwide and represents a risk factor for various chronic inflammatory and neoplastic gastric diseases. In recent years, lipid nanoparticles (LNPs) have demonstrated broad application prospects in vaccine adjuvant research owing to their favorable delivery capacity and immunomodulatory potential. Preparation of LNPs and evaluation of their immunoenhancing effects as adjuvants for a Helicobacter pylori recombinant protein vaccine, as well as their protective efficacy against H. pylori infection.

LNPs were prepared using microfluidic technology, with morphology examined by transmission electron microscopy and particle size and polydispersity index characterized by dynamic light scattering. The activating effects of LNPs on bone marrow-derived dendritic cells (BMDCs) were evaluated in vitro. For in vivo experiments, female BALB/c mice (6 to 8 weeks old, weighing 17 to 20 g) were randomly divided into (n=10): PBS group, UreB/NapA group, and LNP-H+UreB/NapA group. Mice were immunized by intramuscular injection on days 0, 14, and 28. Serum specific antibody titers were measured by ELISA at day 14 after the final immunization, and IFN-γ and IL-17A secretion in mouse spleens was detected by ELISpot. Mice were challenged with H. pylori to establish an infection model. At 4 weeks post-infection, H. pylori colonization in gastric tissue was quantified by probe-based real-time PCR, and adhesion inhibition ability of immunized mice against H. pylori was evaluated through in vitro AGS cell adhesion assay.

The prepared LNPs exhibited uniform morphology, with mean particle size of 100 to 120 nm and polydispersity index of 0.1 to 0.2. LNPs promoted the phagocytosis of antigens by BMDCs and significantly increase the expression levels of costimulatory molecules CD80, CD86, and CD40 on BMDC surfaces (P < 0.001), indicating potential to promote BMDC maturation. LNPs also significantly enhanced the secretions of IL-6 and IL-1β in the supernatants of BMDCs (P < 0.0001). When combined with UreB and NapA, LNPs remarkably elevated the serum titers of specific total IgG, IgG1, and IgG2a (P < 0.05), and promoted the secretions of specific IFN-γ and IL-17A by splenic lymphocytes (P < 0.05), suggesting that LNPs strengthen specific T-cell immune responses. Furthermore, LNPs reduced the colonization of H. pylori in mouse gastric tissues and significantly inhibited the adhesion to gastric mucosal epithelial cells.

LNPs can serve as an effective adjuvant for H. pylori recombinant protein vaccines, effectively enhancing humoral and cellular immune responses and conferring protective effects against H. pylori infection.

To construct lipid nanoparticles (lipopeptide-lipid nanoparticle, LP-LNP) with novel lipopeptides as adjuvants, and initially explore their synergistic effect on mRNA vaccines.

Two novel lipopeptides, SS-10 and SQ18, were designed and synthesized. Microfluidic technology was used to encapsulate lipopeptides in different proportions, as well as mRNAs encoding enhanced green fluorescent protein (eGFP), firefly luciferase (F-luc), and ovalbumin (OVA) into lipid nanoparticles to construct an mRNA delivery system with novel lipopeptides as adjuvants (LP-LNP). The particle size and polydispersity coefficient of LP-LNP were measured using dynamic light scattering. The activation effect on Toll-like receptors 2 (TLR2) was detected using HEK-Blue^TM mTLR2 reporter cells to screen the optimal lipopeptide ratio. The preferred LP-LNP-eGFP-mRNA was transfected into HEK293T cells, and the expression of eGFP was observed under a fluorescence microscope. In vivo imaging was used to investigate the expression level of LP-LNP-F-luc-mRNA in mice. Flow cytometry was used to evaluate the ability of LP-LNP-OVA-mRNA to induce the maturation of dendritic cells (DCs) in draining lymph nodes and cross-presentation of antigens after immunization.

Lipopeptides SQ18 andSS-10 were incorporated into LNP at 0.50% and 0.75% molar ratios, respectively, to obtain LP-LNP with uniform particle size, high encapsulation efficiency, and good in vitro safety. The ability of this formulation to activate TLR2 was significantly stronger than the positive control Pam₂CSK₄ (P<0.01). The preferred LP-LNP obtained effective in vitro transfection, and LP-LNP prepared with SQ18 at 0.50% molar ratio had significantly better in vivo transfection efficiency than traditional LNP (P<0.01), and significantly promoted the maturation of DCs in draining lymph nodes and cross-presentation of antigens (P<0.05).

LP-LNP with novel lipopeptides as adjuvants can enhance the delivery capacity of mRNA and further improve the immune effect of mRNA vaccines.

To investigate the protective effect of Toll-like receptor (TLR) 2/TLR9 agonists, Pam₂ CSK₄ (Pam) and CpG ODN (CpG) on mice infected with Acinetobacter baumannii (Ab) in the lungs.

Female C57 mice (6~8 weeks old) were randomly divided into PBS, Pam, CpG and Pam+CpG groups. In 24 h after intranasal immunization with different doses of the corresponding agonists, the mice were given a lethal dose of Ab infection in the lungs, and the survival rates of the mice were observed. A sublethal dose lung infection model of Ab was then established, and the bacterial colonization in the blood, lungs, liver, kidneys and spleen was measured respectively in the mice after infection. HE staining was used to observe the pathological damages in the lungs and kidneys. The protective effect of the agonists in the immunized mice against Ab was examined at 1, 3 and 7 d after immunization to explore the protective time window. Pam + CpG was used to stimulate A549 cells and RAW264.7 cells to investigate the killing or phagocytic effects on Ab.

Compared to PBS, Pam + CpG treatment significantly improved the survival rate of the mice after a lethal dose of Ab lung infection (P<0.05, P<0.01), reduced bacterial colonization in the blood (P<0.01), lungs (P<0.01), liver (P<0.01), kidneys (P<0.01) and spleen (P<0.01) in the mice after sublethal challenge, and alleviated pathological damage caused by infection. Immunization at 1 or 3 d before infection significantly improved the survival rate (P<0.05), and the protective effect was the best in 3 d after immunization. Furthermore, compared to single PBS, Pam and CpG immunization, Pam+CpG significantly promoted the killing and phagocytic effects of A549 epithelial cells and RAW264.7 cells, respectively, against Ab (P<0.01).

Combined application of TLR2/TLR9 agonists exerts a significant protective effect on both lethal and sublethal infections of Ab, which might be by its promoting the killing or phagocytic effect of lung epithelial cells and macrophages against Ab.

To compare the immune effects of recombinant PKF protein of Acinetobacter baumannii with different immune modes and different adjuvants.

After recombinant PKF protein was obtained by plasmid expression and purification, the protein was then combined with aluminum hydroxide（Al）or with AS03 to immunize mice by intramuscular（im）injection. In another experiment, the combination of PKF and LTK63 or PKF and LP1-34 was adopted to intranasally（in）immunize mice, respectively. The total animals were thus divided into 7 groups: PBS group; PKF group（im）, PKF+Al group（im）, PKF+AS03 group（im）, PKF group（in）, PKF+LTK63 group（in）, and PKF+LP1-34 group（in）. The levels of serum specific IgG and mucosal sIgA in the immunized mice were detected by ELISA. Moreover, the sublethal model of Acinetobacter baumannii pulmonary infection was established, and then the bacterial colonization amount in blood and lung of mice after challenge was measured to evaluate the protective immune efficacy of the recombinant vaccine.

In the intramuscular immunization group, AS03-assisted PKF produced the highest specific IgG antibody level（P<0.01）, and in the nasal immunization group, LTK63 significantly increased the level of specific sIgA in the alveolar lavage fluid（P<0.01）. Bacterial challenge test showed that the bacterial colonization amount was similar in the PKF+AS03（im）group and PKF（im）alone group, while the PKF+LTK63 group（in）obtained remarkably reduced bacterial colonization amount（P<0.01）.

LTK63 assists PKF to better protect against Acinetobacter baumannii pulmonary infection, suggesting that vaccine-mediated mucosal immune response may play a more important role in the infection protection.

To investigate anti-biofilm activity and mechanisms of emodin against methicillin-resistant Staphylococcus aureus (MRSA) in vitro.

Microbroth dilution was explored to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of emodin against MRSA252 strain and clinical isolations. Inhibitory curve were draw to estimate dynamic bacteriostatic effect. Crystal violet staining was used to investigate the inhibitory effect of emodin against MRSA biofilm. The effect of emodin on the morphology of MRSA biofilm was observed by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). The expression of intercellular adhesion (ica) operon was detected by fluorescence quantitative PCR.

The MICs of emodin against different strains of MRSA ranged from 2 to 8 μg/mL, while MBCs were from 4 to 16 μg/mL. Emodin obviously inhibited the formation of biofilm in a dose-dependent manner. Besides, it destroyed biofilm structure and increased the ratio of dead bacteria under biofilm. Fluorescence quantitative PCR showed that emodin significantly down-regulated the expression of ica operon.

Emodin can inhibit MRSA biofilm in vitro, and may restrain the formation of bacterial biofilm by down-regulating the expression of ica operon.