• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2135-2145
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• 2015

VP7, an outer capsid protein of grass carp reovirus (GCRV), was expressed and displayed on the surface of Saccharomyces cerevisiae for developing an efficient vaccine against hemorrhagic disease of grass carp. The result of flow cytometry analysis indicated that protein VP7 could be displayed on the surface of yeast cells after inducing with galactose. The expression of VP7 was confirmed by western blot analysis and further visualized with confocal microscopy. The specific antibodies against VP7 generated from mice were detectable from all immune groups except the control group, which was immunized with untransformed yeast cells. The displaying VP7 on glycosylation-deficient strain EBYΔMnn9 was detected to induce a relatively low level of specific antibody amongst the three strains. However, the antiserum of EBYΔM9-VP7 showed relative high capacity to neutralize GCRV. Further neutralization testing assays indicated that the neutralizing ability of antiserum of the EBYΔM9-VP7 group appeared concentration dependent, and could be up to 66.7% when the antiserum was diluted to 1:50. This result indicates that appropriate gene modification of glycosylation in a yeast strain has essential effect on the immunogenicity of a yeast-based vaccine.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.369-377
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• 1998

Rotavirus is a major cause of severe gastroenteritis in young children and animals throughout the world. The VP7 of rotavirus is thought to induce the synthesis of neutralizing antibodies and to be responsible for determining viral serotypes. The cDNA coding for the VP7 capsid protein of human rotavirus, obtained from Korean patients (HRV-Y14), was cloned and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequences between VP7 of Y14 and that of other foreign isolates showed $92.7~95.2\%$ homology to G1 serotypes (RV-4, KU, K8, WA), $74.2\%$ homolgy to G2 serotype HU-5, $76.4\%$ homology to G3 serotype SA-11, and $77.6\%$ homology to G4 serotype A01321. These data suggest that HRV-Y14 can be classified as a G1 serotype. cDNA coding for VP7 of HRV-YI4 was subcloned into the baculovirus vector and the VP7 glycoprotein was expressed in insect cells. The expressed proteins in Sf9 cell extract and tissue culture fluid were separated on SDS-PAGE, and Western blot analysis with monoclonal antibody raised against the synthetic peptide containing 21 amino acids within the VP7 conserved region was performed. The molecular weight of recombinant VP7 was estimated to be 36 kDa which is about the same size as the native VP7. Addition of tunicamycin in the culture media caused a reduction of the molecular weight of the recombinant VP7 indicating that the expressed protein was glycosylated.

• IMMUNE NETWORK
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• v.4 no.2
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• pp.73-80
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• 2004

Viruses are obligate intracellular parasites which cause infection by invading and replicating within cells. The immune system has mechanisms which can attack the virus in extracellular and intracellular phase of life cycle, and which involve both non-specific and specific effectors. The survival of viruses depends on the survival of their hosts, and therefore the immune system and viruses have evolved together. Immune responses to viral infection may be variable depending on the site of infection, the mechanism of cell-to-cell spread of virus, physiology of the host, host genetic variation, and environmental condition. Viral infection of cells directly stimulates the production of interferons and they induce antiviral state in the surrounding cells. Complement system is also involved in the elimination of viruses and establishes the first line of defence with other non-specific immunity. During the course of viral infection, antibody is most effective at an early stage, especially before the virus enters its target cells. The virus- specific cytotoxic T lymphocytes are the principal effector cells in clearing established viral infections. But many viruses have resistant mechanism to host immune responses in every step of viral infection to cells. Some viruses have immune evasion mechanism and establish latency or persistency indefinitely. Furthermore antibodies to some viruses can enhance the disease by the second infection. Immune responses to viral infection are very different from those to bacterial infection.

• The Journal of the Korean Society for Microbiology
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• v.35 no.1
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• pp.9-18
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• 2000

Immunological mechanisms involving the release of inflammatory factors by HIV-1 infected microglia in the brain have been implicated in the pathogenesis of HIV dementia (HIVD). Since the regulation of matrix metalloproteinases (MMPs) activity can be influenced by variety of inflammatory mediators, this study was undertaken to look for a correlation between the MMP-9 release and the production of TNF-${\alpha}$ in response to HIV-1 p24 in the human monocyte cell line THP-1 as a model for microglia. First, it was shown that HIV-l core p24 antigen induced THP-1 to secrete MMP-9 in a dose response manner while it elicited a little effect on MMP-2 release in human astroglial cell line T98G. Next, it was found that p24 induced THP-1 to secrete TNF-${\alpha}$ without prior differentiation into macrophages by phorbol myristate acetate (PMA) treatment. Furthermore, anti-TNF-${\alpha}$ neutralizing antibodies significantly blocked p24-induced MMP-9 release in a dose dependent manner. Our data indicate that p24 antigen induces monocytic MMP-9 release by triggering up-regulation of TNF-${\alpha}$ secretion.

• Journal of Microbiology
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• v.43 no.6
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• pp.537-545
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• 2005

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used $H-2K^b$ restricted T-cell epitopes of NP. The NP-specific $CD8^+$ T cell response was analyzed using a $^{51}Cr-release$ assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific $CD8^+$ T cell response at eight days after infection. We also found that several different methods to check the NP-specific $CD8^+$ T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited $2\~4$ weeks after immunization and maximized at $6\~8$ weeks. NP-specific $CD8^+$ T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.482-488
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• 2019

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. Replicase (Rep) proteins are considered essential for viral replication. Capsid (Cap) protein is the primary immunogenic protein that induces protective immunity. Little is known about comparison on the immunogenicity of PCV2 Rep and Cap fusion protein and Cap protein. In the present study, recombinant baculoviruses expressing the Rep-Cap fusion protein (Bac-Rep-Cap) and the Cap protein (Bac-Cap) of PCV2 were constructed and confirmed with western blot and indirect fluorescence assay. Immunogenicities of the two recombinant proteins were tested in mice. The titers of antibodies were determined with a PCV2-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The $IFN-{\gamma}$ response of immunized mice was measured by ELISA. The mice immunized with the Bac-Rep-Cap and Bac-Cap successfully produced Cap-specific immunoreaction. The mice immunized with the Bac-Cap developed higher PCV2-specific neutralizing antibody titers than mice injected with the Bac-Rep-Cap. $IFN-{\gamma}$ in the Bac-Rep-Cap group was increased compared to those in the Bac-Cap group. Vaccination of mice with the Bac-Rep-Cap showed significantly decreased protective efficacy compared to the Bac-Cap. Our findings will indubitably not only lead to a better understanding of the immunogenicity of PCV2, but also improved vaccines.

• 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 the Korea Institute of Military Science and Technology
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• v.24 no.3
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• pp.348-355
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• 2021

Hantaan virus(HTNV) causes hemorrhagic fever with renal syndrome(HFRS) with a case fatality rate ranging from <1 to 15 % in human. Hantavax^Ⓡ is a vaccine against the Hantavirus, which has been conditionally approved by the Ministry of Food and Drug Safety(MFDS). However, only 50 % of volunteers had neutralizing antibodies 1 year following the boost. Effective antiviral treatments against HTNV infection are limited. Hantaviruses generally cause asymptomatic infection in adult mice. On the other hand, infection of suckling and newborn mice with hantaviruses causes lethal neurological diesease or persistant infection, which is different from the disease in humans. The development of vaccines and antiviral strategies for HTNV has been partly hampered by the lack of an efficient lethal mouse model to evaluate the efficacy of the candidate vaccines or antivirals. In this report, we established a lethal mouse model for HTNV, which may facilitate in vivo studies on the evaluation of candidate drugs against HTNV. The median lethal dose value of HTNV was calculated by probit analysis of deaths occurring within two weeks. Five groups of ten ICR mice were injected intracranially with serial 2-fold dilutions (from 50 to 3.125 PFU/head) of HTNV. Mice injected with HTNV began to die at 8 days post-infection. The lethal dose required to kill 50 % of the mice (LD₅₀) was calculated to be 2.365 PFU/head.

• Journal of Veterinary Science
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• v.19 no.6
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• pp.817-826
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• 2018

The bursa of Fabricius (BF) is a central humoral immune organ unique to birds. Four bursal peptides (BP-I, BP-II, BP-III, and BP-IV) have been isolated and identified from the BF. In this study, the immunoadjuvant activities of BPs I to IV were examined in mice immunized with H9N2 avian influenza virus (AIV) vaccine. The results suggested that BP-I effectively enhanced cell-mediated immune responses, increased the secretion of Th1 (interferon gamma)- and Th2 (interleukin-4)-type cytokines, and induced an improved cytotoxic T-lymphocyte (CTL) response to the H9N2 virus. BP-II mainly elevated specific antibody production, especially neutralizing antibodies, and increased Th1- and Th2-type cytokine secretion. BP-III had no significant effect on antibody production or cell-mediated immune responses compared to those in the control group. A strong immune response at both the humoral and cellular levels was induced by BP-IV. Furthermore, a virus challenge experiment followed by H&E staining revealed that BP-I and BP-II promoted removal of the virus and conferred protection in mouse lungs. BP-IV significantly reduced viral titers and histopathological changes and contributed to protection against H9N2 AIV challenge in mouse lungs. This study further elucidated the immunoadjuvant activities of BPs I to IV, providing a novel insight into immunoadjuvants for use in vaccine design.

• Journal of Veterinary Science
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• v.21 no.4
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• pp.63.1-63.9
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• 2020

Background: Canine adenovirus type 2 (CAV-2) induces infectious laryngotracheitis in members of the family Canidae, including dogs. To date, no ELISA kits specific for CAV-2 antibody have been commercialized for dogs in Korea. Objectives: We aimed to develop new indirect enzyme-linked immunosorbent assay (I-ELISA) to perform rapid, accurate serological surveys of CAV-2 in dog serum samples. Methods: In total, 165 serum samples were collected from dogs residing in Chungbuk and Gyeongbuk provinces between 2016 and 2018. The Korean CAV-2, named the APQA1701-40P strain, was propagated in Madin-Darby canine kidney cells and purified in an anion-exchange chromatography column for use as an antigen for I-ELISA. The virus-neutralizing antibody titers of CAV-2 in the dog sera were measured by virus neutralization (VN) test. Results: We compared the results obtained between the VN and new I-ELISA tests. The sensitivity, specificity, and accuracy of new I-ELISA were 98.6%, 86.4% and 97.0% compared with VN test, respectively. New I-ELISA was significantly correlated with VN (r = 0.91). Conclusions: These results indicate that new I-ELISA is useful for sero-surveillance of CAV-2 in dog serum.