• Journal of Microbiology
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• v.43 no.2
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• pp.133-143
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• 2005

Shigellosis is a global human health problem. Four species of Shigella i.e. S. dysenteriae, S. flexneri, S. boydii and S. sonnei are able to cause the disease. These species are subdivided into serotypes on the basis of O-specific polysaccharide of the LPS. Shigella dysenteriae type 1 produces severe disease and may be associated with life-threatening complications. The symptoms of shigellosis include diarrhoea and/or dysentery with frequent mucoid bloody stools, abdominal cramps and tenesmus. Shigella spp. cause dysentery by invading the colonic mucosa. Shigella bacteria multiply within colonic epithelial cells, cause cell death and spread laterally to infect and kill adjacent epithelial cells, causing mucosal ulceration, inflammation and bleeding. Transmission usually occurs via contaminated food and water or through person-to-person contact. Laboratory diagnosis is made by culturing the stool samples using selective/differential agar media. Shigella spp. are highly fragile organism and considerable care must be exercised in collecting faecal specimens, transporting them to the laboratories and in using appropriate media for isolation. Antimicrobial agents are the mainstay of therapy of all cases of shigellosis. Due to the global emergence of drug resistance, the choice of antimicrobial agents for treating shigellosis is limited. Although single dose of norfloxacin and ciprofloxacin has been shown to be effective, they are currently less effective against S. dysenteriae type 1 infection. Newer quinolones, cephalosporin derivatives, and azithromycin are the drug of choice. However, fluoroquinolone-resistant S. dysenteriae type 1 infection have been reported. Currently, no vaccines against Shigella infection exist. Both live and subunit parenteral vaccine candidates are under development. Because immunity to Shigella is serotype-specific, the priority is to develop vaccine against S. dysenteriae type 1 and S. flexneri type 2a. Shigella species are important pathogens responsible for diarrhoeal diseases and dysentery occurring all over the world. The morbidity and mortality due to shigellosis are especially high among children in developing countries. A recent review of literature (KotIoff et al.,1999) concluded that, of the estimated 165 million cases of Shigella diarrhoea that occur annually, $99\%$ occur in developing countries, and in developing countries $69\%$ of episodes occur in children under five years of age. Moreover, of the ca.1.1 million deaths attributed to Shigella infections in developing countries, $60\%$ of deaths occur in the under-five age group. Travellers from developed to developing regions and soldiers serving under field conditions are also at an increased risk to develop shigellosis.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.813-819
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• 2019

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe respiratory impairment with a reported mortality rate of ~36% in humans. The absence of clinically available MERS-CoV vaccines and treatments to date has resulted in uncontrolled incidence and propagation of the virus. In vaccine design, fusion with the IgG Fc domain is reported to increase the immunogenicity of various vaccine antigens. However, limited reports have documented the potential negative effects of Fc fusion on vaccine antigens. To determine whether Fc fusion affects the immunogenicity of MERS-CoV antigen, we constructed a Fcassociated MERS-CoV spike protein (eS770-Fc, 110 kDa), whereby human IgG4 Fc domain was fused to MERS-CoV spike protein (eS770) via a Gly/Pro linker using baculovirus as the expression system. For comparative analyses, two eS770 proteins lacking the IgG4 Fc domain were generated using the IdeS protease ($eS770-{\Delta}Fc$) or His tag attachment (eS770-His) and the immunogenicity of the above constructs were examined following intramuscular immunization in mice. Contrary to expectations, non-Fc spike proteins ($eS770-{\Delta}Fc$, eS770-His; 90 kDa) showed higher immunogenicity than the Fc fusion protein (eS770-Fc). Moreover, unlike non-Fc spike proteins, eS770-Fc immunization did not elicit neutralizing antibodies against MERS-CoV. The lower immunogenicity of Fc-fused eS770 was related to alterations in the structural conformation of the spike protein. Taken together, our results indicate that IgG Fc fusion reduces the immunogenicity of eS770 by interfering with the proper folding structure.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.736-744
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• 2023

The introduction of the toxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most Vibrio cholerae strains. Such V. cholerae strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these V. cholerae strains is secreted into the culture medium. In this study, V. cholerae strains that can express intracellular CTB under the control of the toxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the ctxAB promoter to ctxB without ctxA and confirmed CTB expression from the plasmid in V. cholerae containing the toxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7^th to the 20^th amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed V. cholerae strains in which chromosomal ctxAB is replaced by ntrctxB or ntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.

• Genomics & Informatics
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• v.21 no.3
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• pp.42.1-42.23
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• 2023

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Tolllike receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

• Journal of Microbiology and Biotechnology
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• v.34 no.3
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• pp.735-745
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• 2024

Avian influenza is a serious threat to both public health and the poultry industry worldwide. This respiratory virus can be combated by eliciting robust immune responses at the site of infection through mucosal immunization. Recombinant probiotics, specifically lactic acid bacteria, are safe and effective carriers for mucosal vaccines. In this study, we engineered recombinant fusion protein by fusing the hemagglutinin 1 (HA1) subunit of the A/Aquatic bird/Korea/W81/2005 (H5N2) with the Bacillus subtilis poly γ-glutamic acid synthetase A (pgsA) at the surface of Lactobacillus casei (pgsA-HA1/L. casei). Using subcellular fractionation and flow cytometry we confirmed the surface localization of this fusion protein. Mucosal administration of pgsA-HA1/L. casei in mice resulted in significant levels of HA1-specific serum IgG, mucosal IgA and neutralizing antibodies against the H5N2 virus. Additionally, pgsA-HA1/L. casei-induced systemic and local cell-mediated immune responses specific to HA1, as evidenced by an increased number of IFN-γ and IL-4 secreting cells in the spleens and higher levels of IL-4 in the local lymphocyte supernatants. Finally, mice inoculated with pgsA-HA1/L. casei were protected against a 10LD50 dose of the homologous mouse-adapted H5N2 virus. These results suggest that mucosal immunization with L. casei displaying HA1 on its surface could be a potential strategy for developing a mucosal vaccine against other H5 subtype viruses.

• Biomedical Science Letters
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• v.20 no.4
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• pp.250-255
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• 2014

Mycobacterium tuberculosis (MTB) resides and replicates inside macrophages. In our previous report, we reported that CD8+ T cell-mediated immune responses specific for the peptide derived from MTB RNA polymerase beta-subunit ($RpoB_{127-135}$) could be induced in TB patients expressing HLA-$A^*0201$ subtype. In order to examine whether $RpoB_{127-135}$ specific CD8+ T cells can recognize MTB infected macrophages in vitro, CD8+ T cell lines specific for $RpoB_{127-135}$ peptide were generated from peripheral blood mononuclear cells (PBMCs) of healthy HLA-$A^*0201$ subjects by in vitro immunization technique. In this study, we observed $RpoB_{127-135}$ specific CD8+ T cells could recognize and destroy macrophages infected with MTB for 2 to 4 days. $RpoB_{127-135}$ specific CD8+ T cell immune response was inducible from PBMC of healthy subjects expressing HLA-$A^*0206$ subtype, one of HLA-A2 supertype members. Next, we investigated the HLA-I processing mechanism of $RpoB_{127-135}$ peptide in MTB infected macrophages. As a result, the presentation of the MTB derived epitope peptide, $RpoB_{127-135}$, to CD8+ T cells was not inhibited by the treatment with brefeldin-A (ER-Golgi transport inhibitor) or lactacystin (proteasome inhibitor), which blocks the classical HLA-I processing pathway. However, $RpoB_{127-135}$ specific CD8+ T cell activity was blocked either by the blocking agent for the endocytosis (cytochalasin D) or by the blocking antibody (W6/32) for HLA-I molecules. Therefore, the $RpoB_{127-135}$ peptide may be processed by accessing the alternate HLA-I processing pathway. Understanding the processing and presentation mechanisms of the MTB derived proteins will help to improve the efficacy of vaccines and the efficiency of therapeutic agents for TB.

• Journal of Veterinary Clinics
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• v.28 no.3
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• pp.297-302
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• 2011

Avian pathogenic Escherichia coli (APEC) causes a number of extraintestinal diseases in poultry. A virulence factor, P-fimbriae is firmly associated with the diseases. In this study, to develop an effective vaccine for the prevention of APEC, recombinant attenuatted Salmonella Typhimurium vaccines expressing PapA and PapG of P-fimbriae were evaluated whether these induced protective immune responses in murine models. Female BALB/c mice were primed and boosted orally at 7 and 10 weeks of age. In all immunized mice, the antigen-specific serum IgG levels were remained higher than those in the control mice from the fourth week post inoculation till the end of this study. In addition, antigen-specific serum IgG levels in the prime-booster immunized mice were enhanced as compared to the single immunized mice among each immunized group. The antigen-specific mucosal IgA levels in the mice immunized with each strain also induced higher than those in control mice. In addition, serum IgG and fecal IgA levels in mice administered with the combination of both strains were highly induced compared to those in mice immunized with each strain alone. These results indicated that PapA and PapG worked together for inducing high immune responses. To partly discern the nature of immunity induced by the strains, we quantified serum IgG subtypes IgG1 and IgG2a specific to antigens. The PapA and PapG strains biased the immunity to the Th1-type, as determined by the IgG2a/IgG1 ratio. On the other hand, the immunization with the both strains in combination produced mixed Th1- and Th2-type immune responses. These indicated that immunization with the combination of PapA and PapG could elicit both humoral and cell-mediated immunities.