PIRES2-EGFP/CTB-UreI vaccination activated a mixed Th1/Th2/Th17 immune system defense towards Helicobacter pylori infection in the BALB/c mice model.

The occurrence of gastritis, gastric ulcers, distal gastric cancer, and gastric mucosal lymphoma in humans is strongly associated with Helicobacter pylori (H. pylori). Vaccination is an effective preventive measure due to the increasing prevalence of antibiotic resistance. Fusion vaccination is a potentially practical approach. A fusion vaccine was created in this study by combining the cholera toxin B subunit (CTB) with the antigenic H. pylori urease I subunit (CTB-UreI). The CTB-UreI DNA vaccine was chemically cloned into pIRES2-EGFP, and the success of the cloning was validated using PCR and restriction enzyme digestion. An investigation was conducted on the induction of CTB-UreI in Escherichia coli BL21(DE3). The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. The Western blot assay successfully identified the activation of CTB-UreI. In comparison, BALB/c mice receiving pIRES2-EGFP/CTB-UreI vaccination exhibited higher IgG, IgA, IFN-γ, IL-4, and IL-17 levels in their blood samples. In addition, there was a decrease in stomach injuries and bacterial loads. Furthermore, BALB/c mice inoculated with pIRES2-EGFP/CTB-UreI showed a high level of immunity (100%) against the H. pylori challenge. The pIRES2-EGFP/CTB-UreI elicited a combination of Th1/Th2/Th17 immune responses, possibly contributing to an effective defence mechanism. Our data suggests that using this fusion vaccine to prevent H. pylori infection is a promising option.

Bioinformatics-based design of a fusion vaccine with CTLA-4 variable region to combat Brucella

Brucellosis has become a global zoonotic disease, seriously endangering the health of people all over the world. Vaccination is an effective strategy for protection against Brucella infection in livestock in developed countries. However, current vaccines are pathogenic to humans and pregnant animals, which limits their use. Therefore, it is very important to improve the safety and immune protection of Brucella vaccine. In this study, different bioinformatics approaches were carried out to predict the physicochemical properties, T/B epitope, and tertiary structure of Omp2b and Omp31. Then, these two proteins were sequentially linked, and the Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) variable region was fused to the N-terminal of the epitope sequence. In addition, molecular docking was performed to show that the structure of the fusion protein vaccine had strong affinity with B7 (B7-1, B7-2). This study showed that the designed vaccine containing CTLA-4 had high potency against Brucella, which could provide a reference for the future development of efficient brucellosis vaccines.

Selective APC-targeting of a novel Fc-fusion multi-immunodominant recombinant protein (tTax-tEnv:mFcγ2a) for HTLV-1 vaccine development.

AIMS: HTLV-1 causes two life-threatening diseases: adult T-cell leukaemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. Due to the lack of proper treatment, an effective HTLV-1 vaccine is urgently needed. MAIN METHODS: DNA sequences of 11-19 and 178-186 amino acids of HTLV-1-Tax and SP2 and P21 were fused to the mouse-Fcγ2a, or His-tag called tTax-tEnv:mFcγ2a and tTax-tEnv:His, respectively. These constructs were produced in Pichia pastoris, and their immunogenicity and protective properties were assessed in a mouse challenging model with an HTLV-1-MT2 cell line. KEY FINDINGS: The immunogenicity assessments showed significant increase in IFN-γ production in animals receiving tTax-tEnv:mFcγ2a (1537.2 ± 292.83 pg/mL) compared to tTax-tEnv:His (120.28 ± 23.9, p = 0.02). IL-12 production also increased in group receiving tTax-tEnv:mFcγ2a than tTax-tEnv:His group, (23 ± 2.6 vs 1.5 ± 0.6, p = 0.01), respectively. The IFN-γ and IL-12 levels in the Fc-immunised group were negatively correlated with PVL (R = -0.82, p < 0.04) and (R = -0.87, p = 0.05), respectively. While, IL-4 was increased by tTax-tEnv:His (21.16 ± 1.76 pg/mL) compared to tTax-tEnv:mFcγ2a (13.7 ± 1.49, p = 0.019) with a negative significant correlation to PVL (R = -0.95, p = 0.001). SIGNIFICANCE: The mouse challenging assay with tTax-tEnv:mFcγ2a showed 50 % complete protection and a 50 % low level of HTLV-1-PVL compared to the positive control receiving HTLV-1-MT2 (p = 0.001). Challenging experiments for the His-tag protein showed the same outcome (p = 0.002) but by different mechanisms. The Fc-fusion construct induced more robust Th1, and His-tag protein shifted more to Th2 immune responses. Therefore, inducing both T helper responses, but a Th1/Th2 balance in favour of Th1 might be necessary for appropriate protection against HTLV-1 infection, spreading via cell-to-cell contact manner.

B7-1 and GM-CSF enhance the anti-tumor immune effect of DC-tumor fusion vaccine in the treatment of prostate cancer.

The treatment of castration-resistant prostate cancer (CRPC) is always a difficulty in the clinic. Most patients with localized tumor eventually develop CRPC, even if hormone therapy is initially effective. Increasing evidence shows immunotherapy has special advantages compared with traditional therapy in cancer treatment. In this study, we constructed the DC-PC-3 fusion vaccine with B7-1- and GM-CSF-specific modification, and studied its ability to stimulate specific immune response and anti-tumor effect in vitro. The results showed that fusion of DC and tumor cells can improve the expression of associated antigens of DCs. DC-tumor fusion vaccine can strongly promote T cell proliferation and IFN-γ secretion and induce a significant tumor-specific cytotoxic T lymphocyte response. In addition, the B7-1/GM-CSF-modified fusion vaccine showed a more significant anti-tumor effect and greater ability to stimulate the immune response than that without specific modification in vitro. Thus, GM-CSF/B7-1-modified fusion vaccine might be used as a potential therapy strategy for prostate cancer.

A bivalent fusion vaccine composed of recombinant Apx proteins shows strong protection against Actinobacillus pleuroneumoniae serovar 1 and 2 in a mouse model.

Actinobacillus pleuropneumonia (APP) causes porcine pleuropneumoniae, resulting in severe economic losses in the swine industry. Since there are diverse serotypes of APP, it is necessary for vaccines to induce cross-protection. In this report, we developed a bivalent fusion vaccine, the L vaccine composed of ApxIA and ApxIIA fragments. According to the experimental results of the L vaccine, recombinant protein specific-IgG antibody level increased significantly as well as Apx toxin specific-IgG antibody, suggesting toxin-neutralizing effect. Also, the production of both IgG1 and IgG2a indicates this fusion vaccine induces Th1 and Th2 immune reactions. In addition, lymphocytes were proliferated and immune related-cytokines of TNF-α, IL-12, IFN-γ and IL-5 were detected in the serum after the vaccination. The L vaccine showed a perfect cross-protection against APP serovar 1 and 2 that each secrete different Apx exotoxins. These findings reveal that the fusion L vaccine induces specific humoral and cellular immunity, leading to a perfect cross-protection against A. pleuropneumoniae infections in a murine model.

APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice.

Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore, the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine, a combination of an early and a dormant immunogenic protein, ESAT6 and HspX, fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6, a linker, and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6,6-dibehenate(TDB) adjuvant, and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1, Th2, Th17, and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ, 910 pg/mL, and IL-12, 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-ß (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake, inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances, which is important for protection and prevention of damage.

Anti-tumor angiogenesis effect of genetic fusion vaccine encoding murine beta-defensin 2 and tumor endothelial marker-8 in a CT-26 murine colorectal carcinoma model.

Tumor endothelial marker 8 (TEM8) is an endothelial-specific marker that is upregulated during tumor angiogenesis. We previously demonstrated that DNA-based vaccine encoding xenogeneic TEM8 can potentiate anti-angiogenesis immunotherapy of malignancy; nevertheless, it remains to be improved in minimizing immune tolerance. Recently, it has been reported that murine beta-defensin 2 (MBD2) is chemotactic for immature dendritic cells and plays a pivotal role in breaking immune tolerance. Herein, we constructed a genetic fusion vaccine encoding murine TEM8 and MBD2 to investigate whether the novel vaccine preferentially elicits therapeutic antitumor immune responses and suppresses cancerous angiogenesis in mouse models. The anti-angiogenesis effect was determined by microvessel density (MVD) using immunohistochemical staining. The efficacy of the fusion vaccine was primarily assessed by detecting cytotoxic T lymphocyte activity ((51)Cr-release assay). Enzyme-linked immunosorbent spot (ELISpot) assay was used to detect TEM8-specific INF-γ production, and the activity of CTL was further verified by a depletion of CD8(+) T cells via anti-CD8 monoclonal antibody. Our results showed that the DNA fusion vaccine possessed an enhanced therapeutic antitumor immunity through anti-angiogenesis in BALB/c mice inoculated with CT26 cells, and this effect was generally attributed to stimulation of an antigen specific CD8(+) T-cell response against mTEM8. In conclusion, our study demonstrated that the fusion vaccine based on mTEM8 and MBD2 induced autoimmunity against endothelial cells, resulting in deceleration of tumor growth, and could be potential therapeutical application in clinic.

In silico and in vivo studies of truncated forms of flagellin (FliC) of enteroaggregative Escherichia coli fused to FimH from uropathogenic Escherichia coli as a vaccine candidate against urinary tract infections.

The new generation of vaccines against infectious diseases is based on recombinant fusion proteins. Flagellin (FliC) of enteroaggregative Escherichia coli (EAEC) could be considered as a potent adjuvant in designing new vaccines. However, because of its large size, incorporation of this protein with a vaccine antigen might negatively influence recognition of the vaccine epitopes by the immune system. Designing the truncated forms of FliC, capable of inducing innate immune response, enhances the immune responses to the target antigen. We have previously shown that two truncated forms of FliC are able to induce Interleukine-8 production in HT-29 epithelial cell line. In this study we designed recombinant vaccine against urinary tract infections (UTIs) using truncated forms of FliC and type 1 fimbrial FimH adhesin from uropathogenic Escherichia coli (UPEC) and studied their in silico interactions with Toll-like receptor 5 (TLR-5) via docking protocols. The best fusion protein was subjected to cloning and expression. The ability of the recombinant vaccine and the truncated forms in inducing immune responses was investigated. Our results showed that truncated forms are capable of inducing Th1 (forms A and B) and Th2 (form A) responses and fusion vaccine induced strong cellular and humoral immune responses.

Clinical trials of dendritic cell-based cancer vaccines in hematologic malignancies.

The potential for the immune system to target hematological malignancies is demonstrated in the allogeneic transplant setting, where durable responses can be achieved. However, allogeneic transplantation is associated with significant morbidity and mortality related to graft versus host disease. Cancer immunotherapy has the capacity to direct a specific cytotoxic immune response against cancer cells, particularly residual cancer cells, in order to reduce the likelihood of disease relapse in a more targeted and tolerated manner. Ex vivo dendritic cells can be primed in various ways to present tumor associated antigen to the immune system, in the context of co-stimulatory molecules, eliciting a tumor specific cytotoxic response in patients. Several approaches to prime dendritic cells and overcome the immunosuppressive microenvironment have been evaluated in pre-clinical and early clinical trials with promising results. In this review, we summarize the clinical data evaluating dendritic cell based vaccines for the treatment of hematological malignancies.

Improved immunogenicity of fusions between ethanol-treated cancer cells and dendritic cells exposed to dual TLR stimulation.

Tumor-derived transforming growth factor ß1 (TGFß1) generally abrogates the immunogenicity of dendritic cells (DCs) fused to whole cancer cells. We have recently revealed that ethanol-treated neoplastic cells fused to DCs exposed to 2 Toll-like receptor agonists efficiently induce cytotoxic T lymphocytes via TGFß1 blockade and the production of interleukin-12.

Progress in dendritic cell tumor vaccines in cancer immunotherapy / 医学研究生学报

A novel approach of vaccination against cancer is to exploit dendritic cells (DC) as the best antigen presenting cells (APC) and actively immunize cancer patients with a sample of autologous or allogeneic DC primed with tumor antigens. DC vaccination is still at its early stage, however, valuable proofs of concept have been obtained with respect to the capacity of DC to expand cancer directed immune responses. The methods for preparing DC are being improved continuously, and there are many opportunities to improve efficacy at the level of DC biology. An increased number of clinical studies will drive the development of this new area. This paper reviews the production of dendritic cell tumor vaccines and their use in clinical trials, as well as emphasizes some unresolved questions in this immunotherapy.

Effect of Flt-3 ligand on fusion vaccine of dendritic cells and induction of antitumor immunity in vitro / 医学研究生学报

Objective:To investigate the effect of flt-3 ligand on the preparation of the fusional vaccine with dendritic cells and colorectal cancer cells,and the anti-tumor immunological reaction of the vaccine. Methods: ①In the presence of Flt-3 ligand、GM-CSF、IL-4 and TNF,the maturation of dendritic cells was made from peripheral blood;The preparation of the fusional vaccine was made in the presence of PEG. ②The biological characteristics of fusion cells,including their morphological specialties, the expression of cell surface phenotypes, their growth curves, inducing specific cytotoxic T lymphocyte(CTL), their possibility to proliferate into a new carcinoma in nude mice etc, were tested to verify their safety when used as anti-tumor immune reactions. Results: ①Flt-3 ligand can stimulate the full maturation of dendritic cells. Dendritic cells and Lovo cells could be fused by PEG. ②The fusion cells exhibited typical biological characteristics. They could not proliferate into new carcinomas in nude mice, and can inhibit the proliferation of Lovo cells efficiently. Conclusion: Flt-3 ligand can enhance the maturation of dendritic cell. The fused cells had poor proliferation abilities and could not proliferate into new carcinomas in immunodeficient animals. The fusion vaccine could induce specific cytotoxic T lymphocytes in vitro and was safe as a vaccine.

Cytotoxic effects of gastric cancer cell-DC fusion vaccines / 第三军医大学学报

Objective To study on the cytotoxic effects in vivo and in vitro of the T lymphocytes which were activated by the DC-gastric cancer cell fusion vaccines. Methods The mononuclear cells were separated from the peripheral blood of gastric cancer patients and co-cultured with granulocyte-macrophage colony stimulating factors, interleukin-4 and tumor necrosis factor-? to generate mature dendritic cells. The human gastric cancer SGC7901 cells and the dendritic cells were fusioned by using polyethylene glycol, and the pure fusion cells were screened out by culturing with HAT and HT selective culture systems. The mixture of SGC7901 and DC without fusion and the T lymphocytes served as controls. The ability of fusion cells activated by T lymphocytes to kill SGC7901 gastric cancer cells was investigated by MTT in vitro. The anti-tumor effects were evalutated by detecting the growth of the new planted tumors in the nude mice, the apoptosis and proliferation of the planted cancer cells, the pathological changes of the tumor tissues and the prohibitive rate for planted tumors before and after the application of the fusion cell vaccine. Results The mature dendritic cells were obtained from peripheral blood mononuclear cells of gastric cancer patients successfully. Dendritic cells and SGC7901 cells were fusioned and the pure fusion cells were got. Fusion vaccine could induce strong anti-tumor biological effects in vivo and in vitro: Tumor growth was remarkably inhibited and the tumor size in mice receiving fusion cells was (1.298?0.021) cm 3, smaller than that in mice receiving T lymphocytes as treatment (P

The phenotype changes of SGC7901 gastric cancer cell-dendritic cell fusion vaccine / 第三军医大学学报

Objective To obtain SGC7901 gastric cancer cell-dendritic cell fusion vaccines by cell fusion technology. The phenotype changes of the fusion cells were detected to provide experimental data for the following research work. Methods The SGC7901 gastric cancer cells and dendritic cells were induced to be fused by PEG and the pure fusion cells were obtained by selective culture with the HAT/HT culture system. The phenotype changes of the fusion cells were detected by flow cytometer. Results The fusion cells had special morphologic characteristic and the phenotypes of the fusion cells were remarkably higher than the parental dendritic cells. Conclusion The SGC7901 gastric cancer cell-dendritic cell fusion cells kept special morphologic characteristic and their phenotypes were significantly higher than the parental dendritic cells, which may be the cellular base for their stronger biological effects.