Tightly regulated bacteriolysis for production of empty Salmonella Enteritidis envelope

Jawale, C.V.,Kim, S.W.,Lee, J.H. Elsevier Scientific Pub. Co 2014 Veterinary microbiology Vol.169 No.3

To avoid leaky expression of the bacterial host-toxic PhiX174 lysis gene E from the λpR promoter, a convergent promoter construct was made in which gene E was placed between a sense λpR promoter and an anti-sense P<SUB>araBAD</SUB> promoter. In the presence of l-arabinose, leaky transcription of lysis gene E at 28<SUP>o</SUP>C from the sense λpR promoter was repressed by an anti-sense RNA simultaneously expressed from the P<SUB>araBAD</SUB> promoter. The stringent repression of lysis gene E in the absence of induction temperature resulted into higher concentration of bacteria in culture suspension, and consequently higher and stable production of a Salmonella Enteritidis (S. Enteritidis) ghost. The immunogenicity of the S. Enteritidis ghost was evaluated by immunizing chickens. Chickens from the immunized group demonstrated a significant increase in the levels of S. Enteritidis-specific plasma IgG, intestinal sIgA, and lymphocyte proliferative response. After virulent S. Enteritidis challenge, the immunized group exhibited decreased bacterial recovery from organs compared with the non-immunized group. Together, these results demonstrate that the stringent molecular control over leaky transcription of lysis gene E enabled the stable production of S. Enteritidis ghost, and immunization with the S. Enteritidis ghost can protect chickens by inducing robust humoral and cellular immune responses.

Chemically induced bacterial ghosts: a novel approach for advancing biomedical applications

박신영 대한독성 유전단백체 학회 2023 Molecular & cellular toxicology Vol.19 No.4

Background Bacterial ghosts (BGs) are empty cell envelopes derived from bacteria, making them safe and non-replicative, and BGs have shown great potential as a vaccine platform. Specifi cally, chemically induced BGs are generated by selectively removing the cytoplasmic content of bacterial cells while preserving the structural integrity of the cell envelope. Objective Generally, BGs are genetically engineered, but this is limited to Gram-negative bacteria. However, the utilization of chemically induced BGs can be extended to Gram-positive bacteria, resulting in empty bacterial envelopes that hold potential as a platform for drug delivery. Results Chemically induced BGs off er several advantages, including improved safety profi le and immunogenicity, and effi - cient antigen presentation. Preclinical studies have yielded promising results, exhibiting enhanced immune responses and protection against diverse pathogens. Conclusion Chemically induced BGs represent a novel and promising approach for vaccine development, holding the potential for advancing disease prevention and public health. Purpose of review In this review, we discuss key aspects of chemically induced BGs, including their production principles, mechanisms of formation, characterization techniques, immunogenicity, and medical applications. We also discuss the challenges and direction of future research for optimizing production methods for chemically induced BGs, evaluating long-term safety, and undertaking clinical trials to assess their effi cacy.

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

Hajam, Irshad Ahmed,Kim, Je Hyoung,Lee, John Hwa Elsevier 2018 Veterinary immunology and immunopathology Vol.199 No.-

<P><B>Abstract</B></P> <P>The present study aimed to investigate whether the incorporation of flagellin, a TLR5 agonist, in the bacterial ghosts (BGs) of <I>Salmonella</I> 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 <I>E</I> mediated lysis. In this study, the <I>S.</I> 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 <I>S.</I> Gallinarum-flagellin (SG-fliC) ghosts or <I>S.</I> 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-<B>γ</B> responses compared to the chickens vaccinated with the SG ghosts alone. On challenge with the virulent <I>S</I>. 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Flagellin (fliC) in Salmonella Gallinarium (SG) ghosts retain TLR5 binding activity. </LI> <LI> SG-fliC ghosts were completely safe in seven day old layer chicks. </LI> <LI> SG-fliC ghosts mediated early induction of systemic and mucosal antibody responses. </LI> <LI> fliC in SG ghosts enhanced cell mediated immunity and immune protection against fowl typhoid in chickens. </LI> </UL> </P>

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced <i>Vibrio parahaemolyticus</i> Ghosts (VPGs)

Park, Hyun Jung,Oh, Sung,Vinod, Nagarajan,Ji, Seongmi,Noh, Han Byul,Koo, Jung Mo,Lee, Su Hyeong,Kim, Sei Chang,Lee, Ki-Sung,Choi, Chang Won,Piva, Terrence MDPI 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.11

<P>Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene <I>E</I> of bacteriophage PhiX174. In this study, <I>Vibrio parahaemolyticus</I> ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, <I>Limulus amoebocyte</I> lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 10<SUP>6</SUP> CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like <I>Escherichia coli</I> LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1β and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.</P>

Comparison of the immunogenicity between bacterial ghost and formalin-killed bacteria for Vibrio vulnificus

( Se Ryun Kwon ) 한국어병학회 2012 한국어병학회지 Vol.25 No.3

Vibrio vulnificus ghosts (VVG) were generated using a mobilizable vector including a thermosensitive expression cassette by conjugation. The vaccine potential of VVG was investigated in mouse. Mice immunized with VVG showed significantly higher antibody titer than those with formalin-killed V. vulnificus. The present study supports the conceptive usefulness of bacterial ghosts as vaccine candidates.

Comparison of the immunogenicity between bacterial ghost and formalin-killed bacteria for Vibrio vulnificus

Kwon, Se Ryun The Korean Society of Fish Pathology 2012 한국어병학회지 Vol.25 No.3

Vibrio vulnificus ghosts (VVG) were generated using a mobilizable vector including a thermosensitive expression cassette by conjugation. The vaccine potential of VVG was investigated in mouse. Mice immunized with VVG showed significantly higher antibody titer than those with formalin-killed V. vulnificus. The present study supports the conceptive usefulness of bacterial ghosts as vaccine candidates.

Comparison of the immunogenicity between bacterial ghost and formalin-killed bacteria for Vibrio vulnificus

권세련 한국어병학회 2012 한국어병학회지 Vol.25 No.3

Vibrio vulnificus ghosts (VVG) were generated using a mobilizable vector including a thermosensitive expression cassette by conjugation. The vaccine potential of VVG was investigated in mouse. Mice immunized with VVG showed significantly higher antibody titer than those with formalin-killed V. vulnificus. The present study supports the conceptive usefulness of bacterial ghosts as vaccine candidates.

Immunogenicity of a bacterial ghost delivered dengue virus serotype 2 envelope protein domain III followed by an intramuscular protein boosting strategy in mice model

Gayeon Won,Irshad Ahmed Hajam,Perumalraja Kirthika,Eunha Kim,John Hwa Lee 한국예방수의학회 2022 예방수의학회지 Vol.46 No.3

This study aimed to investigate whether bacterial ghosts (BGs), empty cell envelopes of a gram-negative bacterium, delivering envelope protein domain III (EDIII) of dengue virus (DENV) serotype 2 could induce protective immune responses against dengue infection. In this study, we constructed Salmonella Typhimurium BGs expressing and delivering EDIII (BG-EDIII) and evaluated these ghosts for their immunogenicity studies in C57BL/6 mice. Our results demonstrated that the mice vaccinated once orally with BG-EDIII followed by an intramuscular boosting with a recombinant EDIII protein elicited significantly higher humoral and cell-mediated immune responses compared to the BGs alone vaccinated group (p<0.001). Upon challenge with DENV2, significantly lower viral load and liver damage was observed in BG-EDIII vaccinated group than BGs alone control group (p<0.05). The outcomes of this study revealed the ability of BG- EDIII to stimulate immune response with no observable damage to the vital organs.

New typhoid vaccine using sponge-like reduced protocol: development and evaluation

Rehab Bahy 대한백신학회 2023 Clinical and Experimental Vaccine Research Vol.12 No.1

Purpose: Typhoid remains a major health problem, especially in the developing world. Furthermore, the emergence of multidrug-resistant and extensively drug-resistant strains of Salmonella typhi added a sense of urgency to develop more effective typhoid vaccines, one of which is bacterial ghosts (BGs), prepared by both genetic and chemical means. The chemical method includes incubation with numerous agents for a short time at their minimum inhibitory or minimum growth concentrations. This study included the preparation of BGs by a sponge-like reduced protocol (SLRP). Materials and Methods: Critical concentrations of sodium dodecyl sulfate, NaOH, and H2O2 were used. Moreover, high-quality BGs were visualized by scanning electron microscope (SEM). Subculturing was used to confirm the absence of vital cells. Besides, the concentrations of the released DNA and protein were estimated spectrophotometrically. In addition, the integrity of cells was proved by visualizing Gram-stained cells using a light microscope. Furthermore, a comparison between the immunogenicity and safety of the prepared vaccine and the available whole-cell killed vaccine was established. Results: Improved preparation of high-quality BGs of S. typhi, visualized by SEM, revealed punctured cells with intact outer shells. Moreover, the absence of vital cells was confirmed by subculturing. At the same time, the release of respective amounts of proteins and DNA is another evidence of BGs’ production. Additionally, the challenge test provided evidence that the prepared BGs are immunogenic and have the same efficacy as the whole cell vaccine. Conclusion: The SLRP provided a simple, economical, and feasible method for BGs preparation.

Hydrochloric acid-treated Bacillus subtilis ghosts induce IL-1 beta, IL-6, and TNF-alpha in murine macrophage

Kim Young-Min,Lee Kwang-Su,Kim Won-Mun,Kim Min,Park Han-Oh,Choi Chang Won,한중수,Park Shin-Young,Lee Ki-Sung 대한독성 유전단백체 학회 2022 Molecular & cellular toxicology Vol.18 No.2

Background Bacterial ghosts (BGs) are empty cell envelopes commonly generated using Gram-negative bacteria; they represent a potential platform for efficient adjuvant and vaccine delivery systems. However, the efficient production of BGs from bacteria in a short period of time is challenging. Objective The purpose of this study was to investigate the possibility of producing BGs in the Gram-positive Bacillus subtilis using various chemicals, and the potential application of BGs as a novel immunomodulatory agent. Results In this study, Bacillus subtilis ghosts (BSGs) were generated, for the first time to the best of our knowledge, using the minimum inhibitory concentration (MIC) of hydrochloric acid (HCl; 6.25 mg/mL), sulfuric acid (H 2 SO 4 ; 3.125 mg/ mL), and nitric acid (HNO 3 ; 6.25 mg/mL). Among the BSGs generated using these chemicals, HCl-induced BSGs were completely DNA-free as confirmed by real-time polymerase chain reaction. Scanning electron microscopy showed the formation of transmembrane lysis tunnel structures in HCl-induced BSGs. Murine macrophages exposed to the HCl-induced BSGs at a concentration of 1 × 10 5 CFU/mL showed a cell viability of 97.8%. Additionally, HCl-induced BSGs upregulated the expression of pro-inflammatory cytokines including interleukin (IL)-1β, tumor necrosis factor alpha, and IL-6. Furthermore, we found differences in the protein expression profiles between intact live bacteria and BSGs using two-dimensional electrophoresis coupled with peptide mass fingerprinting/matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Conclusion These data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants.