Oncolytic adenoviruses expressing checkpoint inhibitors for cancer therapy.

Despite the remarkable success of immune checkpoint inhibitors (ICIs), primary resistance to ICIs causes only subsets of patients to achieve durable responses due to the complex tumor microenvironment (TME). Oncolytic viruses (OVs) can overcome the immunosuppressive TME and promote systemic antitumor immunity in hosts. Engineered OVs armed with ICIs would likely have improved effectiveness as a cancer therapy. According to the diverse immune cell landscapes among different types of tumors, we rationally and precisely generated three recombinant oncolytic adenoviruses (OAds): OAd-SIRPα-Fc, OAd-Siglec10-Fc and OAd-TIGIT-Fc. These viruses were designed to locally deliver SIRPα-Fc, Siglec10-Fc or TIGIT-Fc fusion proteins recognizing CD47, CD24 or CD155, respectively, in the TME to achieve enhanced antitumor effects. Our results suggested that OAd-SIRPα-Fc and OAd-Siglec10-Fc both showed outstanding efficacy in tumor suppression of macrophage-dominated tumors, while OAd-TIGIT-Fc showed the best antitumor immunity in CD8+ T-cell-dominated tumors. Importantly, the recombinant OAds activated an inflammatory immune response and generated long-term antitumor memory. In addition, the combination of OAd-Siglec10-Fc with anti-PD-1 significantly enhanced the antitumor effect in a 4T1 tumor model by remodeling the TME. In summary, rationally designed OAds expressing ICIs tailored to the immune cell landscape in the TME can precisely achieve tumor-specific immunotherapy of cancer.

A Dendrimer Peptide (KK2DP7) Delivery System with Dual Functions of Lymph Node Targeting and Immune Adjuvants as a General Strategy for Cancer Immunotherapy.

The clinical efficacy of personalized cancer vaccines still needs to be improved due to their insufficient immune effect. The development of innovative adjuvants and lymph node-targeted delivery systems is the key to improving the clinical efficacy of personalized vaccines. However, there is still a lack of an adjuvant delivery system that is simple in preparation and capable of mass production and integrates adjuvant and lymph node targeted delivery functions. Here, this work reports that a simple dendrimer polypeptide (KK2DP7) nanoparticle enhances the immune efficacy of an OVA/neoantigen-based vaccine. Due to its multiple functions as a delivery vehicle, immune adjuvant, and facilitator of dendritic cell migration, KK2DP7 efficiently increases the efficiency of antigen uptake and cross-presentation by antigen-presenting cells (APCs) and delivers antigens to lymph nodes via APCs. Strikingly, the antitumor effect of KK2DP7/OVA is superior to that of commonly used adjuvants such as poly(I:C), CpG, and aluminum adjuvant combined with OVA. Furthermore, KK2DP7/OVA combined with anti-PD-1 antibody is able to prevent tumor recurrence in a postoperative recurrent tumor model. Thus, KK2DP7-based cancer vaccines alone or in combination with immune checkpoint blockade therapies to treat tumors or postoperative tumor recurrence are a powerful strategy to enhance antitumor immunity.

Development of therapeutic vaccines for the treatment of diseases.

Vaccines are one of the most effective medical interventions to combat newly emerging and re-emerging diseases. Prophylactic vaccines against rabies, measles, etc., have excellent effectiveness in preventing viral infection and associated diseases. However, the host immune response is unable to inhibit virus replication or eradicate established diseases in most infected people. Therapeutic vaccines, expressing specific endogenous or exogenous antigens, mainly induce or boost cell-mediated immunity via provoking cytotoxic T cells or elicit humoral immunity via activating B cells to produce specific antibodies. The ultimate aim of a therapeutic vaccine is to reshape the host immunity for eradicating a disease and establishing lasting memory. Therefore, therapeutic vaccines have been developed for the treatment of some infectious diseases and chronic noncommunicable diseases. Various technological strategies have been implemented for the development of therapeutic vaccines, including molecular-based vaccines (peptide/protein, DNA and mRNA vaccines), vector-based vaccines (bacterial vector vaccines, viral vector vaccines and yeast-based vaccines) and cell-based vaccines (dendritic cell vaccines and genetically modified cell vaccines) as well as combinatorial approaches. This review mainly summarizes therapeutic vaccine-induced immunity and describes the development and status of multiple types of therapeutic vaccines against infectious diseases, such as those caused by HPV, HBV, HIV, HCV, and SARS-CoV-2, and chronic noncommunicable diseases, including cancer, hypertension, Alzheimer's disease, amyotrophic lateral sclerosis, diabetes, and dyslipidemia, that have been evaluated in recent preclinical and clinical studies.

Engineering strategies to enhance oncolytic viruses in cancer immunotherapy.

Oncolytic viruses (OVs) are emerging as potentially useful platforms in treatment methods for patients with tumors. They preferentially target and kill tumor cells, leaving healthy cells unharmed. In addition to direct oncolysis, the essential and attractive aspect of oncolytic virotherapy is based on the intrinsic induction of both innate and adaptive immune responses. To further augment this efficacious response, OVs have been genetically engineered to express immune regulators that enhance or restore antitumor immunity. Recently, combinations of OVs with other immunotherapies, such as immune checkpoint inhibitors (ICIs), chimeric antigen receptors (CARs), antigen-specific T-cell receptors (TCRs) and autologous tumor-infiltrating lymphocytes (TILs), have led to promising progress in cancer treatment. This review summarizes the intrinsic mechanisms of OVs, describes the optimization strategies for using armed OVs to enhance the effects of antitumor immunity and highlights rational combinations of OVs with other immunotherapies in recent preclinical and clinical studies.

Enhanced nose-to-brain delivery of siRNA using hyaluronan-enveloped nanomicelles for glioma therapy.

Gliomas are the most malignant brain tumors, and their treatment is very challenging because of the presence of the blood-brain barrier (BBB). Intranasal administration has been considered a noninvasive strategy for glioma therapy in recent years, but our explorations of the intranasal delivery of siRNA-based therapies are still clearly inadequate. In this study, the cell-penetrating peptide DP7-C was enveloped with hyaluronic acid (HA) to develop the multifunctional core-shell structure nanomicelle HA/DP7-C. In vitro studies of HA/DP7-C revealed low cytotoxicity and a higher cell uptake efficiency, which was associated with the interaction between HA and CD44. In vivo experiments indicated that HA/DP7-C delivered the siRNA to the central nervous system through the trigeminal nerve pathway within hours after intranasal administration and that the interaction between HA and CD44 also increased its accumulation at the tumor site. Successful intracellular delivery of an antiglioma siRNA inhibited tumor growth and ultimately prolonged the survival time and decreased the tumor volume in GL261 tumor-bearing mice. In addition, toxicity tests on rats showed no adverse effects on the nasal mucosa and trigeminal nerves. In conclusion, HA/DP7-C is a potential intranasal delivery system for siRNAs in glioma therapy.

Bacteroides fragilis participates in the therapeutic effect of methotrexate on arthritis through metabolite regulation.

Methotrexate (MTX) is a preferred disease-modifying anti-rheumatic drug in the management of rheumatoid arthritis (RA). However, the toxicity and inefficiency of MTX limit its clinical application. Gut microbiota has been implicated in the side effects and efficacy of MTX. In this study, the analysis of the gut microbiota in RA patients revealed that the abundances of intestinal Bacteroides fragilis was reduced after MTX treatment. We observed that MTX has no obvious therapeutic effect in the absence of B. fragilis, while transplantation of B. fragilis restored the efficacy of MTX in antibiotics-pretreated collagen-induced arthritis (CIA) mice. In addition, B. fragilis gavage was accompanied by an increase in butyrate. Supplementation of butyrate restored the response to MTX in gut microbiota-deficient mice, to a similar level achieved by B. fragilis gavage. These results show that gut microbiota-regulated butyrate plays an essential role in the efficacy of MTX, which will provide new strategies to improve the effectiveness of methotrexate in RA treatment.

Lactobacillus reuteri Alleviates Gastrointestinal Toxicity of Rituximab by Regulating the Proinflammatory T Cells in vivo.

Rituximab (RTX) is a widely used anticancer drug with gastrointestinal side effects, such as nausea, vomiting, and diarrhea. The reason for these side effects is still poorly understood. Previous studies have reported that the intestinal microbiota is associated with the occurrence of disease and the therapeutic effect of drugs. In this study, we observed mucosal damage, inflammatory cell infiltration and increased intestinal inflammatory factor expression in RTX-treated mice. RTX also changed the diversity of the intestinal microbiota in mice, and decreased abundance of Lactobacillus reuteri was observed in RTX-treated mice. Further experiments revealed that intragastric administration of L. reuteri in RTX-treated mice attenuated the intestinal inflammatory response induced by RTX and regulated the proportion of helper T (Th) cells. In conclusion, our data characterize the effect of the intestinal microbiota on RTX-induced intestinal inflammation, suggesting that modifying the gut microbiota may represent a positive strategy for managing adverse reactions.

Rational design of innate defense regulator peptides as tumor vaccine adjuvants.

The development of adjuvants has been an empirical process. Efforts to develop a new design and evaluation system for novel adjuvants are not only desirable but also necessary. Moreover, composite adjuvants that contain two or more types of adjuvants to synergistically enhance the immune response are important for adjuvant and vaccine design. Innate defense regulator peptides (IDRs) are promising adjuvants for clinical immunotherapy because they exhibit multifaceted immunomodulatory capabilities. However, the rational design and discovery of IDRs that have improved immunomodulatory activities have been hampered by the lack of screening techniques and the great challenges in the identification of their interaction partners. Here, we describe a screening and evaluation system for IDR design. On the basis of in vitro screening, the optimized IDR DP7 recruited neutrophils, monocytes and macrophages to the site of infection. The adjuvant, comprising the DP7 and CpG oligonucleotide (CpG), induced chemokine/cytokine expression, enhanced the antigen uptake by dendritic cells and upregulated surface marker expression in dendritic cells. Vaccination with the NY-ESO-1 or OVA antigens combined with the adjuvant alum/CpG/DP7 strongly suppressed tumor growth in mice which was due to the improvement of antigen-specific humoral and cellular immunity. Regarding the mechanism of action, GPR35 may be the potential interaction partner of DP7. Our study revealed the potential application of the screening and evaluation system as a strategy for rationally designing effective IDRs or composite adjuvants and identifying their mechanism of action.

Sustained and targeted delivery of siRNA/DP7-C nanoparticles from injectable thermosensitive hydrogel for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers in humans. The inhibition of peptidyl-prolyl cis/trans isomerase (Pin1) gene expression may have great potential in the treatment of HCC. N-Acetylgalactosamine (GalNAc) was used to target the liver. Cholesterol-modified antimicrobial peptide DP7 (DP7-C) acts as a carrier, the GalNAc-siRNA/DP7-C complex increases the uptake of GalNAc-siRNA and the escape of endosomes in hepatocytes. In addition, DP7-C nanoparticles and hydrogel-assisted GalNAc-Pin1 siRNA delivery can effectively enhance the stability and prolong the silencing effects of Pin1 siRNA. In an orthotopic liver cancer model, the GalNAc-Pin1 siRNA/DP7-C/hydrogel complex can potentially regulate Pin1 expression in hepatocellular carcinoma cells and effectively inhibit tumor progression. Our study proves that Pin1 siRNA is an efficient method for the treatment of HCC and provides a sustainable and effective drug delivery system for the suppression of liver cancer.

A Peptide-Based Small RNA Delivery System to Suppress Tumor Growth by Remodeling the Tumor Microenvironment.

MicroRNAs can regulate a variety of physiological and pathological processes and are increasingly recognized as being involved in regulating the malignant progression of cancer, which is an important direction for the study and treatment of cancer. In addition, the tumor microenvironment has gradually become an important direction of study for combating cancer. Researchers can inhibit tumor growth by remodeling and suppressing an immunosuppressive phenotype in the tumor microenvironment. Therefore, the combination of microRNA delivery and tumor microenvironment remodeling may be a potential research direction. In a previous study, we developed a novel cationic and hydrophilic antimicrobial peptide, DP7, by computer simulation. It was found that cholesterol-modified DP7 (DP7-C) has dual functions as a carrier and an immune adjuvant. In this experiment, we used DP7-C to deliver microRNAs or inhibitors intratumorally, where it played a dual role as a carrier and an immune adjuvant. As a delivery vector, DP7-C has more advantages in terms of transfection efficiency and cytotoxicity than Lipo2000 and PEI25K. Components of the DP7-C/RNA complex can effectively escape endosomes after uptake via caveolin- and clathrin-dependent pathways. As an immune adjuvant, DP7-C can activate dendritic cells and promote macrophage polarization. Moreover, it can transform the immunosuppressive tumor microenvironment into an immune-activated tumor microenvironment, indicating its potential as an anticancer therapy. In conclusion, this study identifies a novel microRNA and inhibitor delivery system that can remodel the tumor microenvironment and introduces an alternative scheme for antitumor treatment.

H2O2-Inactivated Salmonella typhimurium RE88 Strain as a New Cancer Vaccine Carrier: Evaluation in a Mouse Model of Cancer.

PURPOSE: This study aimed to describe a novel cancer vaccine developed using H2O2-inactivated Salmonella typhimurium RE88 [with deletions of AroA (the first enzyme in the aromatic amino acid biosynthesis pathway) and DNA adenine methylase] as the carrier. METHODS: The pVLT33 plasmid was used to engineer an RE88 strain induced to express ovalbumin (OVA) by isopropylthiogalactoside (RE88-pVLT33-OVA). The immune responses and anticancer effects of H2O2-inactivated RE88-pVLT33-OVA were compared with those of non-inactivated RE88-pVLT33-OVA and OVA (positive control) in mice carrying OVA-expressing tumors (EG7-OVA) cells. RESULTS: Anti-ovalbumin IgG (immunoglobulin G) titer following vaccination with H2O2-inactivated RE88-pVLT33-OVA was higher for subcutaneous than for intragastric vaccination. When subcutaneous administration was used, H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU (colony forming units)/mouse) achieved an anti-ovalbumin IgG titer higher than that for the same dose of RE88-pVLT33-OVA and comparable to that for 10 µg ovalbumin (positive control). The binding of mouse serum antibodies to EG7-OVA cells was stronger for H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU/mouse) than for 10 µg ovalbumin. Furthermore, subcutaneous vaccination with H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU/mouse) induced greater activation of splenic T cells and more extensive tumor infiltration with CD4+/CD8+ T cells compared with 10 µg ovalbumin (positive control). The mice vaccinated subcutaneously with H2O2-inactivated RE88-pVLT33-OVA at a dose of 2 × 108 or 6 × 108 CFU/mouse had smaller tumors compared with mice in the negative control groups. Tumor weight in mice vaccinated with H2O2-inactivated RE88-pVLT33-OVA at a dose of 2 × 109 CFU/mouse was significantly lower than that in both negative control groups (P < 0.05) and decreased with the increasing dose of H2O2-inactivated RE88-pVLT33-OVA. H2O2-inactivated RE88-pVLT33-OVA was potentially safer than the non-inactivated strain, could carry exogenous antigens, and had specific epitopes that could be exploited as natural adjuvants to facilitate the induction of cellular and humoral immune responses. CONCLUSION: It was anticipated that H2O2-inactivated RE88-pVLT33-OVA could be used as a novel delivery system for new cancer vaccines.

Cholesterol modified DP7 and pantothenic acid induce dendritic cell homing to enhance the efficacy of dendritic cell vaccines.

Dendritic cell (DC)-based cancer vaccines have so far achieved good therapeutic effects in animal experiments and early clinical trials for certain malignant tumors. However, the overall objective response rate in clinical trials rarely exceeds 15%. The poor efficiency of DC migration to lymph nodes (LNs) (< 5%) is one of the main factors limiting the effectiveness of DC vaccines. Therefore, increasing the efficiency of DC migration is expected to further enhance the efficacy of DC vaccines. Here, we used DP7-C (cholesterol modified VQWRIRVAVIRK), which can promote DC migration, as a medium. Through multiomics sequencing and biological experiments, we found that it is the metabolite pantothenic acid (PA) that improves the migration and effectiveness of DC vaccines. We clarified that both DP7-C and PA regulate DC migration by regulating the chemokine receptor CXCR2 and inhibiting miR-142a-3p to affect the NF-κB signaling pathway. This study will lay the foundation for the subsequent use of DP7-C as a universal substance to promote DC migration, further enhance the antitumor effect of DC vaccines, and solve the bottleneck problem of the low migration efficiency and unsatisfactory clinical response rate of DC vaccines.

(EX-4)2-Fc, an effective long-acting GLP-1 receptor agonist, reduces obesity-related inflammation by inhibiting leptin expression.

Obesity has been recognized as a low-grade, chronic inflammatory disease that leads to an increase in obesity-associated disorders, including type 2 diabetes (T2D), fatty liver diseases and cancer. Glucagon-like peptide-1 (GLP-1) is an effective drug for T2D, and it not only has glucose-regulating effects but also has anti-inflammatory effects in obesity. In our previous study, we designed a novel GLP-1 analogue, (EX-4)2-Fc, which has been shown to reduce body weight and improve glucose tolerance in vivo. In this study, we observed that (EX-4)2-Fc also has anti-inflammatory functions in adipose tissue. After the treatment of diet-induced obesity (DIO) mice with (EX-4)2-Fc, we found that the inflammatory response in adipose tissue was significantly attenuated. (Ex-4)2-Fc can reduce obesity-associated proinflammatory cytokine levels and macrophage numbers in DIO mice. In addition, (EX-4)2-Fc treatment resulted in proinflammatory M1-type macrophages beginning to transform into anti-inflammatory M2-type macrophages. The inflammatory mitogen-activated protein kinase (MAPK) signalling pathway and nuclear factor kappa B (NF-κB) were altered in adipose tissue after (EX-4)2-Fc treatment. Leptin has been proven to be closely related to immunity, and we demonstrated that the effect of (EX-4)2-Fc on adipocyte inflammation was related to leptin. The data suggested that (EX-4)2-Fc could modulate the inflammatory response by inhibiting the expression of leptin in adipose tissue.

DP7-C-modified liposomes enhance immune responses and the antitumor effect of a neoantigen-based mRNA vaccine.

To date, many clinical trials have been carried out with neoantigen-specific mRNA vaccines, and positive results have been achieved. However, further improvements in the efficiency of the intracellular delivery of mRNA and the production of a stronger immune response are still worth studying. In this study, we used the cholesterol-modified cationic peptide DP7 (VQWRIRVAVIRK), which was developed in our previous study, with a transmembrane structure and immunoadjuvant function to modify DOTAP liposomes to create a common mRNA delivery system. This system was intended to improve the efficiency of the delivery of mRNA encoding individualized neoantigens to dendritic cells (DCs) and enhance the activation of DCs. The system serves dual functions as a carrier and as an immunoadjuvant. As a carrier of mRNA, DP7-C-modified DOTAP liposomes (DOTAP/DP7-C) could transfer mRNA efficiently into different type of DCs in vitro. As an immunoadjuvant, DOTAP/DP7-C liposomes were shown to be more efficacious in stimulating DC maturation, CD103+ DC (contributing to antigen presentation) production and proinflammatory cytokine secretion than DOTAP liposomes both in vitro and in vivo. In animal studies, the subcutaneous administration of DOTAP/DP7-C/LL2 neoantigen-encoding mRNA complexes significantly inhibited the growth of LL2 in situ and the growth of subcutaneous tumors and stimulated the production of antigen-specific lymphocyte reactions, which were superior to the DOTAP/LL2 neoantigen-encoding mRNA complex group. In conclusion, DOTAP/DP7-C liposomes may serve as a potential universal mRNA delivery system, providing a simple method to increase the efficiency of intracellular mRNA delivery and the immunostimulatory activity of DCs.

Cholesterol-modified DP7 enhances the effect of individualized cancer immunotherapy based on neoantigens.

Personalized cancer vaccines based on neoantigens have become an important research direction in cancer immunotherapy. However, their therapeutic effects are limited by the efficiency of antigen uptake and presentation by antigen presenting cells. Here, the low-toxicity cholesterol-modified antimicrobial peptide (AMP) DP7 (DP7-C), which has dual functions as a carrier and an immune adjuvant, improved the dendritic cell (DC)-based vaccine efficacy. As a delivery carrier, DP7-C can efficiently delivery various antigen peptides into 75-95% of DCs via caveolin- and clathrin-dependent pathways. As an immune adjuvant, DP7-C can induce DC maturation and proinflammatory cytokine release via the TLR2-MyD88-NF-κB pathway and effectively increase antigen presentation efficiency. In addition, DP7-C enhanced the efficacy of DC-based individualized cancer immunotherapy and achieved excellent antitumor effects on mouse tumor models using the OVA antigen peptides and LL2-neoantigens. Excitingly, after DP7-C stimulation, the antigen uptake efficiency of monocytes-derived DCs (MoDCs) in patients with advanced lung cancer increased from 14-40% to 88-98%, the presentation efficiency increased from approximately 15% to approximately 65%, and the proportion of mature MoDCs increased from approximately 20% to approximately 60%. These findings suggest that our approach may be a potentially alternative strategy to produce cancer vaccines designed for individual patients.

Personalized neoantigen-pulsed dendritic cell vaccines show superior immunogenicity to neoantigen-adjuvant vaccines in mouse tumor models.

Development of personalized cancer vaccines based on neoantigens has become a new direction in cancer immunotherapy. Two forms of cancer vaccines have been widely studied: tumor-associated antigen (including proteins, peptides, or tumor lysates)-pulsed dendritic cell (DC) vaccines and protein- or peptide-adjuvant vaccines. However, different immune modalities may produce different therapeutic effects and immune responses when the same antigen is used. Therefore, it is necessary to choose a more effective neoantigen vaccination method. In this study, we compared the differences in immune and anti-tumor effects between neoantigen-pulsed DC vaccines and neoantigen-adjuvant vaccines using murine lung carcinoma (LL2) candidate neoantigens. The enzyme-linked immunospot (ELISPOT) assay showed that 4/6 of the neoantigen-adjuvant vaccines and 6/6 of the neoantigen-pulsed DC vaccines induced strong T-cell immune responses. Also, 2/6 of the neoantigen-adjuvant vaccines and 5/6 of the neoantigen-pulsed DC vaccines exhibited potent anti-tumor effects. The results indicated that the neoantigen-pulsed DC vaccines were superior to the neoantigen-adjuvant vaccines in both activating immune responses and inhibiting tumor growth. Our fundings provide an experimental basis for the selection of immune modalities for the use of neoantigens in individualized tumor immunotherapies.

Prevotella copri is associated with carboplatin-induced gut toxicity.

As a widely used cancer drug, carboplatin often results in serious side effects, such as gut toxicity. In this study, we examined the effects of gut microbiota on mice with carboplatin-induced intestinal mucosal damage. Carboplatin resulted in intestinal mucositis, as indicated by weight loss, diarrhoea, and infiltration of inflammatory cells. It markedly increased the expression of inflammatory cytokines/chemokines in intestine. Carboplatin also altered the diversity and composition of the gut microbiota. A significantly higher abundance of Prevotella copri (P. copri) was observed in carboplatin-treated mice. Moreover, the content of P. copri was positively correlated with the severity of intestinal mucositis. Pretreatment with metronidazole reduced the content of P. copri and relieved the intestinal mucosal injury and inflammation that was induced by carboplatin. Further study revealed that supplementation with P. copri in carboplatin-treated mice resulted in more severe tissue damage, lower tight junction protein expression and higher cytokine expression, and it enhanced both local and systemic immune responses. These data demonstrated that P. copri was involved in the pathological process of carboplatin-induced intestinal mucositis, suggesting a potential attenuation of carboplatin-induced intestinal mucositis by targeting P. copri.

Efficacy of Antimicrobial Peptide DP7, Designed by Machine-Learning Method, Against Methicillin-Resistant Staphylococcus aureus.

Antimicrobial peptides (AMPs) provide a promising strategy against infections involving multidrug-resistant pathogens. In previous studies, we designed a short 12 amino acid AMP DP7, using a machine-learning method based on an amino acid activity contribution matrix. DP7 shows broad-spectrum antimicrobial activities both in vitro and in vivo. Here, we aim to investigate the efficacy of DP7 against multidrug resistant Staphylococcus aureus (S. aureus) and reveal the potential mechanisms. First, by measuring the killing kinetics of DP7 against S. aureus and comparing these results with antibiotics with different antimicrobial mechanisms, we hypothesize that DP7, in addition to its known ability to induce cell wall cation damage, can also exert a full killing effect. With FITC-conjugated or biotin-labeled DP7, we tracked DP7's attachment, membrane permeation and subsequent intracellular distribution in S. aureus. These results indicated that the possible targets of DP7 were within the bacterial cells. Transcriptome sequencing of S. aureus exposed to DP7 identified 333 differentially expressed genes (DEGs) influenced by DP7, involving nucleic acid metabolism, amino acid biosynthesis, cell wall destruction and pathogenesis, respectively, indicating the comprehensive killing efficacy of DP7. In addition, the genome sequencing results of the induced DP7 resistant strain S. aureus DP7-R revealed two-point mutations in the mprF and guaA gene. Moreover, in a murine model for MRSA blood stream infection, intravenously treating mice with DP7 showed a good protective effect on mice. In conclusion, DP7 is an effective bactericide for S. aureus, which deserves further study for clinical application and drug development.

Contribution of sex­based immunological differences to the enhanced immune response in female mice following vaccination with hepatitis B vaccine.

Hepatitis B virus (HBV) vaccination is regarded as the most economical and effective method for the prevention and control of HBV infection, a major global health problem. Previous studies have suggested that there may be sex­specific differences regarding the immune response to the HBV vaccine in humans; however, the mechanisms associated with these sex­specific differences are yet to be elucidated. In the present study, sex­based immunological differences in mice following HBV vaccination were investigated to determine the mechanisms underlying sexual dimorphism, with the aim of identifying potential targets for clinical intervention. Balb/c mice (n=6) were vaccinated intramuscularly on 3 different days (days 0, 14 and 28) with the HBV vaccine. Sera were analyzed via ELISA for the presence of HBV surface antigen (HBsAg)­specific immunoglobulin G (IgG), and of different IgG subtypes, 3 weeks following the third injection. Enzyme­linked immunosorbent spot assays were conducted to determine interleukin­4/interferon­Î³ secretion. Immunological memory stimulated by the vaccine was detected via flow cytometry analysis and ELISA 1 week following the booster immunization. The seroconversion of the treated female group was higher compared with the male group at one week following the second vaccination. Female mice exhibited significantly increased HBsAg antibody titers compared with males at 1­5 weeks following the third vaccination. Sera obtained from vaccinated female mice exhibited markedly increased titers of IgG1 and IgG2b compared with those from male mice. Furthermore, female mice exhibited elevated cytotoxic T lymphocyte responses and immune memory. Collectively, the results of the present study indicated that sex­based immunological differences affected the dynamics and characteristics of the immune response in mice immunized with the HBV vaccine.

Hydrogen peroxide-inactivated bacteria induces potent humoral and cellular immune responses and releases nucleic acids.

The problem of nosocomial infection is seriously escalating. Bacterial vaccines are indispensable for preventing infections caused by multi-drug resistant organisms. Some researchers have put forward the use of hydrogen peroxide (H2O2) as a new technology platform for virus deactivation. This deactivated virus can induce the number of CD8+ T lymphocytes, which can enhance antiviral responses. Although, H2O2 treatment has been rarely reported on the exploration of bacterial deactivation, H2O2 deactivation of whole-cell bacteria could be a potential novel approach for bacterial vaccine development. Here we present a strategy for H2O2-deactivated bacterial whole-cell vaccines, for two major pathgens, Pseudomonas aeruginosa and Staphylococcus aureus. The proactive effects of vaccination were assessed in vitro and in vivo. H2O2-deactivation of bacterial vaccines retains more complete epitopes and exhibits lower toxicity, as compared to formaldehyde, a conventional deactivator that was investigated in this study. Furthermore, H2O2-deactivated bacterial vaccines induce anti-infection responses through enhancement of humoral immunity and cellular immunity. Vaccination with H2O2-deactivated whole-cell bacteria in mice mainly elicits whole-cell specific antibody titers and balances the IgG2a and IgG1 response, predominantly with IgG3 induction at the later stages, meanwhile provides opsonic protection against challenge with pathogens. Finally, H2O2 deactivation of bacteria has been found to cause the release of bacterial DNA which is followed by NF-κB activation. These findings demonstrate that the deactivation of whole-cell bacteria with H2O2 is potentially advantageous for immune responses. Considering the prevention of drug-resistant infections, this deactivation method could be simultaneously applied as an innovative strategy for bacterial vaccine development.