• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.982-986
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• 2009

For the production of ghost bacteria vaccine to prevent the streptococcal disease in aquaculture fish species, a double cassettes vector was constructed and cloned in Escherichia coli DH5${\alpha}$. Ghost bacteria vaccine production from Escherichia coli DH5${\alpha}$/pHCE-InaN-GAPDH-Ghost 37 SDM (SIG) was maximized at a glucose concentration of 1 g/l, agitation of 300 rpm, and aeration of 1 vvm. The maximal efficiency of ghost bacteria formation was obtained at the mid-exponential phase ($OD_{600}=2.0$) with the concentration of 0.77 g/l for SIG. The molecular mass of GAPDH was detected at 67 kDa with the insoluble fraction, by SDS-PAGE and Western blot. The protective efficacy of ghost bacteria vaccine was evaluated by challenge test using olive flounder. The cumulative mortalities of the positive control, formalin-killed cell (FKC) vaccine, and SIG vaccine immunized groups were 91%, 74%, and 57%, respectively. These results suggest that SIG vaccine showed efficacy as a vaccine and had a higher potential to induce protective antibodies than did FKC vaccine.

• Journal of fish pathology
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• v.25 no.3
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• pp.159-164
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• 2012

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.

• Journal of Life Science
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• v.19 no.7
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• pp.845-851
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• 2009

A vector harboring double cassettes; a heterologous gene expression cassette of pHCE-InaN-antigen and a ghost formation cassette of pAPR-cI-E lysis 37 SDM was constructed and introduced to E. coli DH5a. For the production of a bacterial ghost vaccine, bacterial ghosts from E. coli / Streptococcus iniae with four different types of antigens - enolase, GAPDH, sagA and piaA - were produced by the optimization of fermentation parameters such as a glucose concentration of 1 g/l, agitation of 300 rpm and aeration of 1 vvm. Efficiency of ghost bacteria formation was evaluated with cultures of OD$_{600}$=1.0, 2.0 and 3.0. The efficiency of the ghost bacteria formation was 99.54, 99.67, 99.99 and 99.99% with inductions at OD$_{600}$=3.0, 1.0, 2.0 and 1.0 for E. coli/S. iniae antigens enolase, piaA, GAPDH and sagA, respectively. Ghost bacteria as a vaccine was harvested by centrifugation. The antigen protein expressions were analyzed by SDS-PAGE and western blot analysis, and the molecular weights of the enolase, piaA, GAPDH and sagA were 78, 26, 67 and 26 kDa, respectively. The molecular weights of the expressed antigens were consistent with theoretical sizes obtained from the amino acid sequences.

• Journal of Life Science
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• v.24 no.1
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• pp.39-45
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• 2014

Salmonella typhimurium MMP13 and S. typhimurium ${\chi}8554$ were derived from weak JOL401 and strong ${\chi}3339$ virulent strains. Heat-labile subunit B (LT-B) was used as an adjuvant to increase the effectiveness of the vaccine. Plasmid pMMP184 carrying a ghost cassette was transformed into MMP13 and ${\chi}8554$ to produce the ghost, and the prepared ghost cells were administered into the muscles of BALB/c mice. In the absence of the adjuvant, the total IgG content showed a tendency to increase contrary to the original virulent strength. In contrast, in the presence of the adjuvant, the strain that originated from the strong virulent showed a tendency to promote the immune more than that of weak virulent strain. However, the final concentration of total IgG was similar between the compared groups, indicating that the originated virulent strength does not affect a specific immune. Other elements of the immunoglobulins IgG1, IgG2a, and sIgAs did not show a specific trend. The results of Salmonella challenge showed a similar tendency to regardless of the originated virulence. Taken together, the results suggest that the Salmonella ghost cells promoted the immune system of BALB/c mice, irrespective of the virulence applied to create the ghost cells.

• Journal of Life Science
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• v.25 no.11
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• pp.1197-1203
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• 2015

Salmonella Typhimurium (ST) JOL389 and χ3339 are strong virulent strains against mouse. ST χ8554 is derived by deletion of the asd gene from ST χ3339. Plasmid pMMP184 carrying a ghost cassette was transformed into ST χ8554, and ST χ8554 ghost cells were prepared and administrated via the oral route to BALB/c mice. Change in the amount of total IgG was not elicited to boosting of single ST χ8554 ghost cells, but the content was increased from 6 weeks after the 3^rd administration. However, when the ST JOL389 ghost cells is administered together with ST χ8554 ghost cells, the content of total IgG was increased in 2 weeks post primary administration. It was found that the content of total IgG of the group mixed with ST JOL389 ghost cells showed an increased value of 8 times or more at 10 weeks when compared with the group of ST χ8554 ghost cells. The content of IgG1, IgG2a, and sIgA in both groups increased from 4 weeks postprimary administration. As a challenge test of virulent ST χ3339, χ8554 (pMMP184) and χ8554 (pMMP184)/JOL389 ghost cell groups showed protection of 50% or more when compared to the control group. These results suggest that the preparation of combined ghost cells from a strong virulent ST increases immunity more than a single strain.

• Journal of Life Science
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• v.28 no.4
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• pp.496-507
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• 2018

Immunization has been performed for centuries and is generally accepted as a sustainable method of controlling bacteria, viruses, and mediated and infectious diseases. Despite many studies having been performed on animal subjects to demonstrate the importance of toxin immunity, the use of toxoid vaccines in humans and animals has been limited for a long time. Recently, the development of the toxoid antigen delivery system has been facilitated using novel nano-medicinal technology. The micro/nano-carrier has been used to improve vaccination coverage as well as reduce vaccine costs. A micro/nano-carrier is a micro/nano-sized material that delivers immune cargo, including recombinant or peptide toxoid antigens. These toxoid antigens are either encapsulated in the interior or displayed on the surface of micro/nano-carriers as a way to protect them from the cellular machinery. In particular, the combination of toxoid antigens and micro/nano-carriers can induce phagocytosis through the specific interactions between GCs and macrophages; thus, the toxoid antigens can be delivered easily into the macrophages. This paper reviews recent achievements of micro/nano-carriers in the field of vaccine delivery systems such as microbial ghost cells (GCs, Bacterial ghost cells and Yeast ghost cells), gene-manipulated outer membrane vesicles (OMVs) and biocompatible, polymer-based nanoparticles (NPs, NP-Carrier and NP-Cage). Finally, this review shows various aspects in terms of the hosts' immune responses.

• Clinical and Experimental Vaccine Research
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• v.12 no.1
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• pp.70-76
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• 2023

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.

• Korean Journal of Veterinary Service
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• v.43 no.2
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• pp.89-97
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• 2020

This study was carried out to examine a novel inactivated Salmonella Typhimurium (S. Typhimurium) vaccine candidate for protection of mice against salmonellosis by immunization of BALB/c mice using various type adjuvant. The novel type-inactivated vaccine candidate was constructed by adding Chlorhexidine digluconate solution. BALB/c mice were divided into 6 groups of 15 mice apiece. The mice were intramuscularly (IM) primed at 6 weeks of age and were IM boosted 8 weeks of age. Groups A and B mice were injected with sterile phosphate-buffered saline as controls; group C mice were inoculated with 5×10⁸ cells/100 µL of formalin-inactivated S. Typhimurium cells and adjuvant ISA70; groups D~F mice were immunized with 5×10⁸ cells/100 µL of the inactivated vaccine candidate and adjuvant ISA70, adjuvant IMS1313 and adjuvant IMS1313 containing 30 ㎍/mL of GI24, respectively. All mice (except group A mice) were orally challenged with a virulent S. Typhimurium strain at 10 weeks of age. Mice from groups C-F had significantly increased IgG levels compared to control groups (A-B) mice. The levels of splenocyte IFN-γ and IL-4 in mice of all groups were measured by ELISA, resulting in increased immunity in group F mice compared to those of groups A-E mice. These data suggested that systemic and cell-mediated immune responses were highly induced by IM immunization with the vaccine candidate and adjuvant IMS1313 containing GI24. Furthermore, clinical signs such as death were observed in only 20% of group F mice after virulent Salmonella strain challenge, however, groups B and C (100%), and groups D and E (60%) mice died. This data suggested that mice immunized by intramuscular prime and booster with this vaccine candidate and adjuvant IMS1313 containing GI24 effectively protected mice from salmonellosis.

• Journal of Life Science
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• v.24 no.2
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• pp.154-160
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• 2014

Ghost cells have been recognized as eliciting humoral and cell-mediated immune responses and have also been predicted to play a role as an immune adjuvant. In this study, we used the intramuscular (IM) route to inject BALB/c mice with four vaccine groups constructed from Salmonella typhimurium ghost (STG) cells originating from different virulent strains and complex STG groups instead of heat-labile toxin (LT)-B, a type of adjuvant. Although the complex STG groups exhibited a response after a short delay, the groups showed final total IgG levels similar to those of the LT-B group, which encodes LT-B from pMMP300. The IgG1 response to the ${\chi}$3339 group was the highest response at 6 weeks, whereas IgG2a responses to the ${\chi}$3339 and JOL389 groups were higher at 6 and 8 weeks compared to those of the LT-B group. The response of vaginal sIgA to the LT-B group was generally higher than that of the other groups, whereas fecal sIgA to the LT-B group exhibited lower responses. Protection to virulent S. typhimurium in all groups was above 80%, which was similar to the LT-B group. Taken together, we suggest that STG complex groups can be used as an immune adjuvant instead of LT-B.

• Journal of Life Science
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• v.31 no.8
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• pp.755-760
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• 2021

Swine diarrhea is a livestock disease that causes huge economic losses to pig farms. In general, diarrhea occurs because of the proliferation of pathogenic Escherichia coli (E. coli). The toxins produced by the proliferated E. coli cause edema in pigs. Although the proliferation of these coliforms can be prevented by using a vaccine, the vaccines containing chemically produced dead bacteria are not very effective, making it difficult to control the proliferation of E. coli. Therefore, there is a need to develop new, more effective vaccines. In this study, we prepared killed F4+ and F18ab+ E. coli, which induce diarrhea and edema in pigs, using the extracts of immune-induced silkworms containing antimicrobial peptides and examined their availability as a killed-bacteria vaccine. First, the antimicrobial activity analysis of the prepared immune-induced silkworm extract was conducted using the radial diffusion assay. The results showed high activity against both F4+ and F18ab+ E. coli. The production efficiency of E. coli dead cells was determined using the colony-counting method. The concentration of the E. coli dead cells was the highest (50 mg/ml) when treated at 4℃. In addition, the analysis of the prepared dead cells using a transmission electron microscope confirmed that E. coli leaked out of the cytoplasm and the cell membrane remained intact. Therefore, F4+ and F18ab+ E. coli produced using immune-induced silkworms extract are considered to be highly available as bacterial ghost vaccines that can help prevent swine diarrhea and the resulting edema.