Bacterial Ghosts Research Articles

Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis

Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis

Bacterial ghost as delivery vehicles loaded with DNA vaccine induce significant and specific immune responses in common carp against spring viremia of carp virus

Although vaccine is a prophylaxis measure for the contagious and fatal disease caused by spring viremia of carp virus (SVCV), until now, no commercial vaccine is available for worldwide use. To find more effective and safe means for the control of SVCV infection, here, an Escherichia coli DH5α bacterial ghost (DH5α-BG, non-living bacterial) based DNA vaccine delivery system (BG/pEG-G) was constructed. Carp were injected intramuscularly by different treatments (PBS, pEGFP, BG, pEG-G and BG/pEG-G), and the immunoprotective effect induced by these treatments were analyzed. We found a stronger and longer duration immune response (serum antibody production and immune-related genes expression) can be induced in fish vaccinated with BG/pEG-G in comparison with those vaccinated with pEG-G alone. Additionally, a good immune protective effect (relative percentage survival >81.48%) was observed in fish vaccinated with BG/pEG-G vaccine at the dose of 0.2 μg DNA/g of fish. Overall, this study for the first time demonstrates the ability of DH5α-BG as DNA vaccine carrier against SVCV infection in carps, suggesting a practical multivalent strategy for the control of aquatic disease.

An oral double-targeted DNA vaccine induces systemic and intestinal mucosal immune responses and confers high protection against Vibrio mimicus in grass carps

Vibrio mimicus (V. mimicus) is an enteric pathogen causing serious vibriosis in aquatic animals, and intestinal mucosal immunity elicited by oral DNA vaccines plays a vital role in the resistance to the pathogen. Yet, the immunogenicity of oral naked DNA vaccine is poor due to the degradation in the digestive tract. Therefore, it is necessary to use targeted DNA delivery strategy for improving the potency of vaccine. In our previous studies, the epitopes of OmpU and VMH proteins from V. mimicus have been identified. Here, a double-targeted DNA vaccine based on the multi-epitope peptide (named OVepis) of V. mimicus was developed by using E. coli DH5α bacterial ghosts (BGs) and invariant chain-like protein (Iclp) of grass carps as exogenous and endogenous targeting delivery carriers, respectively, and then the efficacy was evaluated in grass carps via oral gavage administration. The results showed that the loading efficiency and capacity of DH5α-BGs to pcDNA3.1-Iclp-OVepis were 89.91% and 16.6 μg per milligram BGs, respectively. DH5α-BGs loaded with pcDNA3.1-Iclp-OVepis targeted efficiently RAW264.7 cells and mediated effective gene expression. Higher transcription levels of the Iclp-OVepis gene were detectable in the different tissues of fish in the double-targeted DNA vaccine (DH5α-BGs/pcDNA3.1-Iclp-OVepis) group than that in the endogenous-targeted DNA vaccine (pcDNA3.1-Iclp-OVepis) group at 21 dpv. The double-targeted DNA vaccine significantly enhanced SOD activity, LZM activity and C3 contents in the serum and intestinal mucus of immunized fish. Specific antibody levels in the serum and intestinal mucus were markedly increased in the double-targeted DNA vaccine group compared to the other DNA vaccine groups. at each examined time point. Likewise, the proliferative activities of peripheral blood and intestinal intraepithelial lymphocytes of fish in the double-targeted DNA vaccine group were also significantly higher at 28 dpv. Importantly, the vaccine conferred high protection against V. mimicus challenge with 81.11% RPS. This study suggested that the oral double-targeted DNA vaccine might be used as a promising vaccine candidate to control V. mimicus infection.

Bacterial Ghosts Carrying 5-Fluorouracil: A Novel Biological Carrier for Targeting Colorectal Cancer.

Bacterial ghosts (BGs) are non-deformed bacterial cell envelopes that possess undamaged external configurations for precise attachment to different cells of the human body. The Escherichia coli BGs were successfully produced using a modified sponge-like reduced protocol and characterized by SEM. Four different concentrations of 5-fluorouracil (5-FU) were used to study the impact on the "ghosts" cell wall. 5-FU was then loaded into the BGs and the loading capacity (LC %) and entrapment efficiency (EE %) were determined and were found to be 38.3 ± 0.8 and 76.6 ± 0.8, respectively. The in vitro release studies were conducted in dialysis bags over a time period of 16days and the accumulative 5-FU released (%) was calculated. Overall, 69.2% of the ghost-associated 5-FU was released from the BGs and release from the E. coli ghosts is governed by non-Fickian diffusion. The Caco-2 cell line was used to investigate the cytotoxicity of 5-FU-loaded BGs.

Use of a modified bacterial ghost lysis system for the construction of an inactivated avian pathogenic Escherichia coli vaccine candidate

Vaccination is an effective strategy to prevent avian colibacillosis. Bacterial ghosts (BGs) are prepared by the controlled expression of the phiX174 gene E, which mediates the lysis of Gram-negative bacteria. Staphylococcal nuclease A may be used to produce BGs for further inactivation of host bacteria and elimination of residual genetic material. In this study, the double promoter lysis plasmid (pUC19-ΔcI857-E-rrnB-pL-SN) was successfully constructed and BGs were prepared at 37 °C. The cleavage efficiency of Escherichia coli BGs was 99.9%. Furthermore, to evaluate the immunological effects of the BG vaccines in chickens, a BG vaccine was prepared using the serotype O2 avian pathogenic Escherichia coli deletion strain (DE17ΔluxSΔaroA). The results showed that the BG vaccine was able to achieve over 90% immune protection against virulent challenge using the same serotype O2 strain (DE17 or CE35), while it showed poor cross-protection against serotypes O1 and O78 (data not shown). The enzyme-linked immunosorbent assay results showed that the antibody levels in the immunized groups were higher than in the control group (p < 0.05), with the BG group being the highest. The cytokine tests showed that the levels of interferon-γ in the BG immune group were higher than in the phosphate-buffered saline (PBS) control group (non-immune) (p < 0.01) and the formalin-inactivated vaccine immune group (p < 0.05), and the levels of tumor necrosis factor-α in the BG group were higher than in the formalin-inactivated vaccine (p > 0.05) and the PBS control groups (p < 0.05). In addition, pathological analysis revealed that the PBS control group showed typical fibrinous pericarditis and perihepatitis, whereas the immune group showed no obvious pathological changes. In summary, our findings provide a new strategy for the prevention and control of avian colibacillosis.

Mannheimia haemolytica in bovine respiratory disease: immunogens, potential immunogens, and vaccines.

Mannheimia haemolytica is the major cause of severe pneumonia in bovine respiratory disease (BRD). Early M. haemolytica bacterins were either ineffective or even enhanced disease in vaccinated cattle, which led to studies of the bacterium's virulence factors and potential immunogens to determine ways to improve vaccines. Studies have focused on the capsule, lipopolysaccharide, various adhesins, extracellular enzymes, outer membrane proteins, and leukotoxin (LKT) resulting in a strong database for understanding immune responses to the bacterium and production of more efficacious vaccines. The importance of immunity to LKT and to surface antigens in stimulating immunity led to studies of individual native or recombinant antigens, bacterial extracts, live-attenuated or mutant organisms, culture supernatants, combined bacterin-toxoids, outer membrane vesicles, and bacterial ghosts. Efficacy of several of these potential vaccines can be shown following experimental M. haemolytica challenge; however, efficacy in field trials is harder to determine due to the complexity of factors and etiologic agents involved in naturally occurring BRD. Studies of potential vaccines have led current commercial vaccines, which are composed primarily of culture supernatant, bacterin-toxoid, or live mutant bacteria. Several of those can be augmented experimentally by addition of recombinant LKT or outer membrane proteins.

Preparation of porcine enterotoxigenic Escherichia coli (ETEC) ghosts and immunogenic analysis in a mouse model

Enterotoxignenic Escherichia coli (ETEC)-associated colibacillosis causes high levels of morbidity and mortality in neonatal piglets. Vaccination is among effective strategy to fight against ETEC-related diseases. Bacterial ghosts (BGs) are empty bacterial envelopes, which substain subtle antigenic comformation in bacterial outer membrane. In this study, a BG vaccine was generated using porcine ETEC isolated strain DQ061 and evaluated its safety and immunogenicity in a mouse model. The recombinant bacteria were constructed by transformation of lysis plasmid pHH43 and generation of BGs was conducted in a lysis rate of 99.93% by incubation of the recombinant bacteria at 42 °C for 2 h. Mice were immunized subcutaneously twice in 2-week intervals with BGs, BGs emulsified with ISA 206 adjuvant, or formalin-inactivated ETEC vaccine after safety test. Mice with either of two BG vaccines developed higher titer of antibodies, secreted higher titer of interleukin 4, gamma interferon and alpha tumor necrosis factor after 2 doses than those with formalin-inactivated ETEC vaccine or those with adjuvant placebo (P < 0.01). The quantity of CD4+ and CD8+ T lymphocyte in spleen was higher in both BG groups than that in the inactivated vaccine group or adjuvant group 2 weeks post boost immunization (P < 0.05). The vaccinated mice were challenged intraperitoneally with 10 × LD50 dose of DQ061. Mice with the BGs plus adjuvant were completely protected against challenge, compared to 60% protection of mice with the inactivated vaccine. Mice exhibited decreased tissue lesion and reduced bacterial loads in the BGs groups by comparison with those with the inactivated vaccine or adjuvant only. Our results validated that the ETEC BGs bear high safety and immunogenicity in a mouse model, suggesting a potential of further evaluation in a pig model.

Generation and immunity effect evaluation of biotechnology-derived Aeromonas veronii ghost by PhiX174 gene E-mediated inactivation in koi (Cyprinus carprio koi)

Aeromonas veronii is a conditional pathogen causing high mortality in many freshwater fish species worldwide. Bacterial ghosts are nonliving Gram-negative bacteria devoid of cytoplasmic contents, which induce protective immunity against microbial pathogens. The aims of this study were: a) to produce A. veronii ghost (AVG) constructed by PhiX174 gene E; b) to evaluate the specific, non-specific immune effects and protective immunity of AVG against A. veronii in koi. The lysis plasmid pBBR-E was constructed by cloning PhiX174 gene E into the broad-host-range vector pBBR1MCS2, and then transformed into A. veronii 7231. AVG was generated by increasing the incubation temperature up to 42 °C. Lysis of A. veronii occurred 3 h after temperature induction and completed in 12 h. The efficiency of ghost induction was 99.9998 ± 0.0002%. Koi were immunized intraperitoneally with AVG, formalin-killed bacteria (FKC) or phosphate buffered saline (PBS) respectively, and then respiratory burst (RB), myeloperoxidase (MPO), lysozyme (LZM), malondialdehyde (MDA), complement 3 (C3) and antibody activities were examined in serum. Compared with negative control of PBS, the RB, MPO, LZM activities were significantly higher in koi immunized with AVG (P < 0.05). Nevertheless, the MDA activities of AVG treatment were significantly lower than those of PBS treatment (P < 0.05). The serum agglutination titers and IgM antibody titers in AVG group were significantly higher than those in FKC or PBS groups. After challenged with the parent strain A. veronii 7231, the average mortality of AVG group was significantly lower than that of FKC and PBS groups (P < 0.05) and the relative percent survival (RPS) of AVG group (73.92%) was higher than that of FKC group (43.48%). Therefore, AVG have the potential to induce protective immunity and they may be ideal vaccine candidates against A. veronii in koi.

Generation of a novel Streptococcus agalactiae ghost vaccine and examination of its immunogenicity against virulent challenge in tilapia

Streptococcus agalactiae (S. agalactiae) is a gram-positive pathogen that causes a wide range of infections in fish and other animals including humans. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and are well represented as novel vaccine candidates. In this study, we examined the immunogenicity and protective efficacy of S. agalactiae ghosts (SAG) against a virulent challenge in tilapia. Nonliving SAG was generated by a culture with Penicillin and Streptolysin, and then treated with the MIC of sodium hydroxide. The formation of a transmembrane lysis tunnel structure in SAG was visualized by electron microscopy. To investigate the SAG as a vaccine candidate, fish were divided into three groups, A (SAG immunized), B [Formalin-inactivated S. agalactiae (FSA) immunized] and C (phosphate-buffered saline, PBS-immunized control). The IgM antibody responses were significantly stronger in the SAG-immunized group than in FSA-immunized group, which was higher than in the non-immunized control group (P < 0.05). Moreover, phagocytic activity (percent phagocytes, PP) was significantly higher (p < 0.05) in the SAG-immunized group than in FSA-immunized group, which was higher than in the non-immunized control group (P < 0.05). In addition, non-specific immune immunity, such as lysozyme and superoxide dismutase activities, in the SAG-immunized fish showed significantly higher activities than FSA-immunized fish and the control group fish (P < 0.05). Also, fish immunized with SAG and FSA showed significantly higher (p < 0.05) gene expression of IL-1β, TNF-α, IFN-γ and TGF-β in the head kidney and spleen than fish treated with PBS during the whole observed period. In addition, fish immunized with SAG showed significantly higher gene expression of L-1β, TNF-α, and TGF-β in the spleen than in the FSA-immunized fish. Although there was no significant (P > 0.05) difference of survival rate (SR) or relative percent survival (RPS) between SAG and FSA immunized groups, they were all significantly more protected against the S. agalactiae challenge (SR: 86.67%, RPS: 76.395) and (SR: 80.00%, RPS: 67.50%) respectively, compared to the PBS-treated group (SR: 33.33%). These results suggest that immunization with SAG induces immune responses and provides protection against a virulent S. agalactiae challenge.

Different concentrations of Edwardsiella tarda ghost vaccine induces immune responses in vivo and protects Sparus macrocephalus against a homologous challenge

Bacterial ghosts (BGs) can be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria. They are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. Here we prepared Edwardsiella tarda ghost (ETG) and tested different concentrations in vaccination trials. The results showed that serum IgM antibody titers were significantly higher in three different concentration immunization groups than control group (P < 0.05), However, there was no significant (P > 0.05) difference between the immunized groups. The phagocytic percentage (PP) was significantly higher (P < 0.05) in ETG immunized groups than in the control group from 3 days post-treatment. The PP continued to rise with time until day 21, when the values of three ETG immunized groups were 45.7%,51.2% and 50.7%, respectively. In addition, phagocytic index (PI) was significantly higher (P < 0.05) in ETG immunized groups than in the control group after 7 days post-treatment. However, there was no significant (P > 0.05) difference of PP or PI between immunized groups. In addition, non-specific immune immunity, such as acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme activities displayed a similar pattern in all immunized groups, all immunized fish showed significantly higher activities than control group fish (P < 0.05). Most importantly three ETG immunized groups were all significantly more protected against the E. tarda challenge (19/25, 76% survival), (21/25, 84% survival) and (20/25, 80% survival) respectively, compared to (9/25, 36% survival) survival in the control group, but there was no significant (P > 0.05) difference of survival rate (SR) or relative percent survival (RPS) between immunized groups. All these results suggest that an ETG could stimulate cellular and humoral immunity, and could be used as a vaccine candidate in S.m. In summary, ETG can protect fish from Edwardsiellosis, and there is no significant difference in SR and RPS when three different concentrations of ETG are used, so it can easily be developed as a vaccine for mechanical and artificial operations.

Design and immune characterization of a novel Neisseria gonorrhoeae DNA vaccine using bacterial ghosts as vector and adjuvant

Gonorrhea, an important sexually transmitted disease, is becoming a growing public health problem around the globe. Vaccination is considered the best long-term approach for control of infection. In this study, we designed a novel Neisseria gonorrhoeae (N. gonorrhoeae) DNA vaccine delivered by bacterial ghosts and characterized its immune responses in vitro and in vivo. Our results demonstrate that bacterial ghosts greatly promoted BMDCs maturation and activation. Bacterial ghosts loaded with N. gonorrhoeae DNA vaccine were efficiently taken up by mouse macrophage RAW264.7 cells. Furthermore, oral immunization with the ghost vaccine candidate elicited greater CD4+ and CD8+ T cell responses and induced higher IgG responses than N. gonorrhoeae DNA vaccine alone. In addition, mice immunized with the vaccine candidate responded with a significant rise in bactericidal antibody titer. These results suggest that bacterial ghosts may function as a vaccine adjuvant by promoting BMDCs maturation, which in turn enhances the immune responses to the vaccine antigens. This study also highlights the potential of using bacterial ghosts as antigen delivery system in the development of an efficacious gonorrhea vaccine.

Tumor lysate-loaded Bacterial Ghosts as a tool for optimized production of therapeutic dendritic cell-based cancer vaccines

Cancer immunotherapy with dendritic cell (DC)-based vaccines has been used to treat various malignancies for more than two decades, however generally showed a limited clinical success. Among various factors responsible for their modest clinical activity is the lack of universally applied, standardized protocols for the generation of clinical-grade DC vaccines, capable of inducing effective anti-tumor immune responses. We investigated Bacterial Ghosts (BGs) – empty envelopes of Gram-negative bacteria – as a tool for optimized production of DC vaccines. BGs possess various intact cell surface structures, exhibiting strong adjuvant properties required for the induction of DC maturation, whereas their empty internal space can be easily filled with a source tumor antigens, e.g. tumor lysate. Hence BGs emerge as an excellent platform for both the induction of immunogenic DC maturation and loading with tumor antigens in a single-step procedure. We compared the phenotype, cytokine secretion profile, functional activity and ability to induce immunogenic T-cell responses in vitro of human monocyte-derived DCs generated using BG platform and DCs matured with widely used lipopolysaccharide (LPS) plus interferon-γ cocktail and loaded with tumor lysate. Both approaches induced DC maturation, however BG-based protocol was superior to LPS-based protocol in terms of the ability to induce DCs with a lower tolerogenic potential, resulting in a more robust CD8+ T cell activation and their functional activity as well as significantly lower induction of regulatory T cells. These superior parameters are attributed, at least in part, to the ability of BG-matured DCs to resist potential immunosuppressive and pro-tolerogenic activity of various tumor cell lysates, including melanoma, renal carcinoma and glioblastoma.

Incorporation of membrane-anchored flagellin into Salmonella Gallinarum bacterial ghosts induces early immune responses and protection against fowl typhoid in young layer chickens

The present study aimed to investigate whether the incorporation of flagellin, a TLR5 agonist, in the bacterial ghosts (BGs) of Salmonella Gallinarum can enhance protective immune responses against fowl typhoid, a septicemic disease of poultry, in chickens. BGs are empty cell envelopes derived from Gram-negative bacteria through the bacteriophage phiX174 gene E mediated lysis. In this study, the S. Gallinarum ghosts carrying flagellin were genetically constructed utilizing a lysis plasmid pJHL184-flagellin, designed for the coexpression of the flagellin and the lysis protein E. The adjuvant effect of flagellin was evaluated by immunizing seven day old brown nick layer chicks once orally with either S. Gallinarum-flagellin (SG-fliC) ghosts or S. Gallinarum (SG) ghosts alone. Our results showed that immunization with the SG-fliC ghosts elicited early and higher systemic (IgG) and mucosal (IgA) antibody responses compared to the SG ghosts alone, although not always statistically significant. Flow cytometric analysis of the CD3 + CD4+ and the CD3 + CD8+ T cell populations in peripheral blood mononuclear cells were higher in chickens immunized with the SG-fliC ghosts compared to the chickens vaccinated with the SG ghosts alone. Furthermore, the chickens immunized with SG-fliC ghosts exhibited significantly (p < 0.05) higher IL-6 and IFN-γ responses compared to the chickens vaccinated with the SG ghosts alone. On challenge with the virulent S. Gallinarum wild type strain at 28th day post immunization, 5 of 10 birds died (50%) in case of SG-fliC ghost group while 60% (6 of 10 birds died) mortality was observed in the SG ghost group. Collectively, these results suggest that the expression of flagellin in SG ghosts improves antigen-specific humoral and cell mediated immune responses, and can enhance protective efficacy of the BG-based vaccines against the virulent challenges.

Bacterial ghost of avian pathogenic E. coli (APEC) serotype O78:K80 as a homologous vaccine against avian colibacillosis

Avian Colibacillosis is among the major causes of economic loss in the poultry industry worldwide, with a more vivid impact on developing countries. The involvement of several bacteria has made it challenging to develop effective vaccines for this disease, particularly because it is notoriously difficult to make a vaccine that contains all the contributing pathogenic bacteria. Here, we report the design and fabrication of a bacterial ghost (BG) of E. coli O78:K80, which is among the major bacterial serotypes responsible for this disease. The generated ghost is then exploited as a homologous vaccine against Avian Colibacillosis. We demonstrate that hole formation in the cell wall of E. coli O78:K80 can happen properly in optical densities as high as 0.8 compared to the 0.3–0.4 standard for bacteria like E. coli TOP10. This is especially advantageous for mass production of this ghost which is a vital factor in development of any BG-based vaccine. Compared to E. coli TOP10, we faced a great challenge in transforming the wild type bacteria with the E-lysis plasmid which was probably due to higher thickness of the cell wall in O78:K80. This, however, was addressed by treating the cell wall with a different combination of ions.The vaccine was administered to Ross 308 broiler chickens via injection as well as through their respiratory system at a dose of 1010 BGs, repeated 3 times at weekly intervals. Chickens were then challenged with the wild type O78:K80 at a dose of 1011 bacteria together with Infectious Bronchitis H120 vaccine (as immunosuppressant) one week after the last immunization. Air sac lesions were significantly reduced in BG vaccinated groups in comparison with the control group. The levels of IFNγ, IgA and IgY were measured in the serum of immunized chickens as an indication of immune response and were compared with those of the chickens vaccinated with killed bacteria. The results show that O78:K80 BG can be used as an efficient homologous vaccine against Colibacillosis disease in poultry. We expect our findings can serve as the starting point for designing more sophisticated vaccines that contain all three major pathogenic bacteria involved in avian Colibacillosis.

Enhanced anti-proliferative efficacy of epothilone B loaded with Escherichia coli Nissle 1917 bacterial ghosts on the HeLa cells by mitochondrial pathway of apoptosis

Epothilones constitute a new class of microtubule-stabilizing anti-cancer agents with promising preclinical and clinical activity. However, its systemic application still causes some toxic side effects. To reduce these undesired effects, advanced drug delivery systems based on cell targeting carriers are needed currently. In this study, the high quality bacterial ghosts of the probiotic Escherichia coli Nissle 1917 (EcN) were prepared in a large scale and retained fully intact surface structures for specific attachment to mammalian cells. The EcN ghosts could be efficiently loaded with the low hydrophilic drug Epothilone B (Epo B) and the maximal load efficiency was approximately 2.5% (w/w). Cytotoxicity assays revealed that Epo B-ghosts exhibited enhanced anti-proliferative properties on the HeLa cells. The Epo B associated with EcN ghosts was more cytotoxic at least 10 times than the free Epo B at the same concentrations. Apoptosis assays showed that both Epo B-ghosts and free Epo B induced time course-dependent apoptosis and necrosis in HeLa cells, respectively. While the former induced more apoptosis and necrosis than the latter. Furthermore, the cytochrome C release and the activation of caspase-3 were more remarkable after treatment with the Epo B-ghosts compared to the free Epo B, which implied that Epo B-ghosts might more effectively induce the apoptosis mediated by mitochondrial pathway in HeLa cells. Therefore, the higher anti-proliferative effects of the Epo B-ghosts on the HeLa cells were mediated by mitochondrial pathway of apoptosis. The EcN ghosts may provide a useful drug delivery carrier for drug candidates in cancer therapy.

Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis

ABSTRACTColorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality worldwide. At late stage of the disease CRC often shows (multiple) metastatic lesions in the peritoneal cavity which cannot be efficiently targeted by systemic chemotherapy. This is one major factor contributing to poor prognosis. Oxaliplatin is one of the most commonly used systemic treatment options for advanced CRC. However, drug resistance – often due to insufficient drug delivery – is still hampering successful treatment. The anticancer activity of oxaliplatin includes besides DNA damage also a strong immunogenic component. Consequently, the aim of this study was to investigate the effect of bacterial ghosts (BGs) as adjuvant immunostimulant on oxaliplatin efficacy. BGs are empty envelopes of gram-negative bacteria with a distinct immune-stimulatory potential. Indeed, we were able to show that the combination of BGs with oxaliplatin treatment had strong synergistic anticancer activity against the CT26 allograft, resulting in prolonged survival and even a complete remission in this murine model of CRC carcinomatosis. This synergistic effect was based on an enhanced induction of immunogenic cell death and activation of an efficient T-cell response leading to long-term anti-tumor memory effects. Taken together, co-application of BGs strengthens the immunogenic component of the oxaliplatin anticancer response and thus represents a promising natural immune-adjuvant to chemotherapy in advanced CRC.

Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects.

Vibrio alginolyticus (V. alginolyticus) is a common pathogen for humans and marine aquatic animals. Vibriosis of marine aquatic animals, caused by V. alginolyticus, has become more prevalent globally in recent years. Hence, a safe and effective vaccine is urgently needed for the control of this disease. Here, the strain 16‐3 isolated from the large yellow croaker (Larimichthys crocea) suffered from canker was identified as V. alginolyticus based on morphological, biochemical, and 16S rDNA sequencing analysis. Then, recombinant temperature‐controlled lysis plasmid pBV220‐lysisE was electroporated into the strain 16‐3 to generate V. alginolyticus bacterial ghosts (VaBGs) by inducing lysis gene E expression, and the safety and immune effects of VaBGs were further investigated in mice and large yellow croaker. The results showed that VaBGs were as safe as formalin‐killed V. alginolyticus cells (FKC) to mice and fish. Compared with FKC and PBS groups, significant elevations of the serum agglutinating antibody titer, serum bactericidal activity, lymphocyte proliferative responses, and levels of four different cytokines (Th1 type: IL‐2, TNF‐α; Th2 type: IL‐4 and IL‐6) in serum were detected in the VaBGs group, indicating that a Th1/Th2‐mediated mixed immune response was elicited by the VaBGs. More importantly, after challenged with the parent strain 16‐3, VaBGs‐vaccinated mice and fish showed higher protection than FKC‐vaccinated mice, the relative percent of survival (RPS) being 60%, 66.7% and 40%, respectively. Taken together, this is the first demonstration that the newly constructed V. alginolyticus ghosts may be developed as a safe and effective vaccine against V. alginolyticus infection in aquaculture.

A Salmonella Typhi ghost induced by the E gene of phage φX174 stimulates dendritic cells and efficiently activates the adaptive immune response.

Previously, we genetically engineered a Salmonella Typhi bacterial ghost (STG) as a novel inactivated vaccine candidate against typhoid fever. The underlying mechanism employed by the ghost in stimulating the adaptive immune response remains to be investigated. In this study, we aimed to evaluate the immunostimulatory effect of STG on mouse bone marrow-derived dendritic cells (BMDCs) and its activation of the adaptive immune response in vitro. Immature BMDCs were stimulated with STG, which efficiently stimulated maturation events in BMDCs, as indicated by upregulated expressions of CD40, CD80, and major histocompatibility complex class II molecules on CD11+ BMDCs. Immature BMDCs responded to STG stimulation by significantly increasing the expression of interleukin (IL)-6, which might indicate the induction of dendritic cell maturation in vivo (p < 0.05). In addition, ghost-stimulated murine BMDCs showed significant expressions of interferon gamma and IL-4, which can drive the development of Th1 and Th2 cells, respectively, in co-cultured CD4+ T cells in vitro. These results suggest that STG can effectively stimulate maturation of BMDCs and facilitate subsequent immune responses via potent immunomodulatory cytokine responses.

Construction of Salmonella Pullorum ghost by co-expression of lysis gene E and the antimicrobial peptide SMAP29 and evaluation of its immune efficacy in specific-pathogen-free chicks

In this study, a safety enhanced Salmonella Pullorum (S. Pullorum) ghost was constructed using an antimicrobial peptide gene, and evaluated for its potential as a Pullorum disease (PD) vaccine candidate. The antimicrobial peptide SMAP29 was co-expressed with lysis gene E to generate S. Pullorum ghosts. No viable bacteria were detectable either in the fermentation culture after induction of gene E- and SMAP29-mediated lysis for 24 h or in the lyophilized ghost products. Specific-pathogen-free (SPF) chicks were intraperitoneally immunized with ghosts at day 7 of age and no mortality, clinical symptoms or signs of PD such as anorexia, depression and diarrhea were observed. On challenge with a virulent S. Pullorum strain at 4 wk post-immunization, a comparatively higher level of protection was observed in the S. Pullorum ghost immunized chickens with a minimum of pathological lesions and bacterial loads compared to the birds in inactivated vaccine groups. In addition, immunization with the S. Pullorum ghosts induced a potent systemic IgG response and was associated with significantly increased levels of cytokine IFN-γ and IL-4 and relative percentages of CD4+ and CD8+ T lymphocytes. Our results indicate that SMAP29 can be employed as a new secondary lethal protein to enhance the safety of bacterial ghosts, and to prepare a non-living bacterial vaccine candidate that can prevent PD in chickens.

A novel protocol for bacterial ghosts’ preparation using tween 80

Bacterial ghosts (BGs) can be prepared by both genetic and chemical means. Genetic method include using lysis gene E. Chemical method include incubation with numerous agents for a short time at their minimum inhibitory or minimum growth concentrations (MIC or MGC). The aim of this study is to prepare the BGs with a new protocol via exposing the bacterial cells to tween 80 for an extended period of time followed by sudden reduction of the surrounding pH. Salmonella enterica serovar typhimurium ATCC 13311 was used for this purpose. The cells were incubated in 7% v/v tween 80 solution in Muller-Hinton broth for 24 h at 37 °C then pH was decreased to 3.6 by adding lactic acid for one hour. The bacterial pellets were separated by high speed centrifugation, and then washed three times by half normal saline solution. High quality BGs were visualized by scanning electron microscopy (SEM) revealing punctured cells with intact outer shells and at least one intramembranous tunnel. The absence of vital cells was confirmed by subculturing. The release of respective amounts of proteins and DNA is another evidence of ghost’s production. In addition, the integrity of cells was proved by visualization of Gram-stained cells using light microscopy. In conclusion, this new protocol is simple, economic and feasible for BGs preparation.