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Kim, Myung Hee,Kang, Jung-Ok,Kim, Joo-Young,Jung, Hi Eun,Lee, Heung Kyu,Chang, Jun ELSEVIER 2019 ANTIVIRAL RESEARCH Vol.163 No.-
<P><B>Abstract</B></P> <P>Nucleoprotein is highly conserved among each type of influenza viruses (A and B) and has received significant attention as a good target for universal influenza vaccine. In this study, we determined whether a recombinant adenovirus encoding nucleoprotein of type B influenza virus (rAd/B-NP) confers protection against influenza virus infection in mice. We also identified a cytotoxic T lymphocyte epitope in the nucleoprotein to determine B-NP-specific CD8 T-cell responses. We found that B-NP-specific CD8 T cells induced by rAd/B-NP immunization played a major role in protection following influenza B virus infection using CD8 knockout mice. To assess the effects of the administration routes on protective immunity, we immunized mice with rAd/B-NP via intranasal or intramuscular routes. Both groups showed strong NP-specific humoral and CD8 T-cell responses, but only intranasal immunization provided complete protection against both lineages of influenza B virus challenge. Intranasal but not intramuscular administration established resident memory CD8 T cells in the airway and lung parenchyma, which were required for efficient protection. Furthermore, rAd/B-NP in combination with rAd/A-NP protected mice against lethal infection with both influenza A and B viruses. These findings demonstrate that rAd/B-NP could be further developed as a universal vaccine against influenza.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We develop a novel and universal influenza B vaccine based on recombinant adenovirus expressing nucleoprotein (NP). </LI> <LI> Vaccination is capable of inducing B-NP-specific humoral and cellular immune responses. </LI> <LI> This protection correlates with the establishment of resident memory CD8 T cells in the lungs. </LI> <LI> rAd/B-NP immunization combined with rAd/A-NP provides protection against both influenza A and B strains. </LI> </UL> </P>
( Sung-jin Yoon ),( Young-jun Park ),( Hyun Ju Kim ),( Jinwoo Jang ),( Sang Jun Lee ),( Sunwoo Koo ),( Moo-seung Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.10
Accurate and rapid diagnosis of influenza infection is essential to enable early antiviral treatment and reduce the mortality associated with seasonal and epidemic infections. Immunochromatography is one of the most common methods used for the diagnosis of seasonal human influenza; however, it is less effective in diagnosing pandemic influenza virus. Currently, rapid diagnostic kits for pandemic influenza virus rely on the detection of nucleoprotein (NP) or hemagglutinin (HA). NP detection shows higher specificity and is more sensitive than HA detection. In this study, we time-dependently screened expression conditions, and herein report optimal conditions for the expression of recombinant nucleoprotein (rNP), which was 48 h after infection. In addition, we report the use of the expressed rNP in a rapid influenza diagnostic test (SGT i-flex Influenza A&B Test). We constructed expression vectors that synthesized rNP (antigen) of influenza A and B in insect cells (Sf9 cells), employed the purified rNP to the immunoassay test kit, and clearly distinguished NPs of influenza A and influenza B using this rapid influenza diagnostic kit. This approach may improve the development of rapid test kits for influenza using NP.
Fathalla, O.A.,Gad, H.S.M.,Maghaby, A.S. The Pharmaceutical Society of Korea 2000 Archives of Pharmacal Research Vol.23 No.2
In continuation of the previous work (Fathalla, 1992) on the synthesis of some heterocycles containing uracil moiety, we report herein the incorporation of uracil moiety into cyan-opyridine thione, thiosemecarbazone, semicarbazone, cyanopyridine, ami nocyano pyridine, isoxazoline, pyrazoline, pyrimidine, triazolo pyrimidine, pyran, selena and thiazole derivatives which might modify their biological activities. The biological studies revealed that the chemical compound III f showed high molluscicdal activity than other compounds. The profile of the nucleoprotein extracted from chemically (compound IIIc, e, f and g) treated or UV-irradiated B.alexandrina snails did not show appreciable differences when compared to non-treated (native) snails by using SDS-PAGE, where no obvious qualitative or quantitative differences were observed. Immunization of experimental animals with the nucleoprotein extracted from native, chemically (compound III f & g) treated or physically treated B.alexandrina snails induced significant protection against challenge with normal S.mansoni cercariae, as compared to the non-immunized challenged control. As well as , a decrease in the number of granuloma formation and the size range of granuloma was also observed in immunized animals. It is concluded that, compounds III f and g have a potent molluscicidal activity. They also induced chemical modification comparable to that induced by physical treatment in the snail's nucleoprotein, which could possibly be used in immunization against S. mansoni infection.
Improved Immune Response to Recombinant Influenza Nucleoprotein Formulated with ISCOMATRIX
( Cargnelutti Diego E. ),( Maria V. Sanchez ),( Paula Alvarez ),( Lorena Boado ),( Graciela Glikmann ),( Nora Mattion ),( Eduardo A. Scodeller ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.3
Current influenza vaccines elicit antibodies effective against homologous strains, but new strategies are urgently needed for protection against emerging epidemic or pandemic strains. Although influenza vaccine candidates based on the viral nucleoprotein (NP) or matrix protein do not elicit sterilizing immunity, they have the advantage of inducing immunity that may cover a larger number of viral strains. In this study, recombinant NP produced in Escherichia coli was purified and formulated in combination with the adjuvant ISCOMATRIX. This formulation increased a NP-specific immunity in mice, with a Th1 profile, and may constitute a promising low-cost influenza vaccine candidate, with ability to stimulate humoral and cellular immune responses.
( Nguyen Hong Phuong ),( Chaewon Kwak ),( Chang-kyu Heo ),( Eun Wie Cho ),( Jihyun Yang ),( Haryoung Poo ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.5
Influenza, which is a highly contagious disease caused by the influenza A virus, continues to be a major health concern worldwide. Although the accurate and early diagnosis of influenza virus infection is important for controlling the spread of this disease and rapidly initiating antiviral therapy, the current influenza diagnostic kits are limited by their low sensitivity. In this study, we developed several new influenza nucleoprotein (NP)-specific monoclonal antibodies (mAbs) and compared their sensitivity and specificity of those with commercially available anti-NP mAbs. Three mAbs, designated M24.11, M34.3, and M34.33, exhibited higher reactivities to recombinant NPs and A/Puerto Rico/8/1934 (H1N1) viral lysates compared with the commercial mAbs, as assessed using enzyme-linked immunosorbent assays. M34.3 and M34.33 showed higher reactivities with A/California/04/09 (pandemic H1N1) and A/ Philippines/2/82 (H3N2) viral lysates than the commercial mAbs. In contrast, M24.11 had marked reactivity with H3N2 but not with pandemic H1N1. Immunofluorescent confocal microscopy showed that the three mAbs effectively detected the presence of influenza virus in lung tissues of mice infected with A/Puerto Rico/8/1934. These results indicate that the newly developed M34.3 and M34.33 mAbs could be useful for the development of influenza diagnostics.
이소영,강정옥,장준 대한백신학회 2019 Clinical and Experimental Vaccine Research Vol.8 No.1
Purpose: The influenza B virus diverges into two antigenically distinct lineages: B/Yamagata and B/Victoria. Influenza B is the dominant circulating virus during some influenza seasons, and recent data demonstrated that influenza A and B infection similarly cause severe clinical symptoms in hospitalized patients. Nucleoprotein (NP) is a good target for a universal influenza vaccine. This study investigated whether NP epitope variation within two lineages affects the dominant cytotoxic T lymphocyte (CTL) responses induced by vaccination and the resultant protective immunity. Materials and Methods: The NP of B/Yamagata/16/1988, the representative strain of the Yamagata lineage, includes a dominant CTL epitope, FSPIRITFL, while B/Shangdong/7/1997 from the Victoria lineage has one amino acid difference in this sequence, FSPIRVTFL. Two recombinant replication-deficient adenovirus (rAd)-vectored vaccines expressing either NP were prepared (rAd/B-NP(I) and rAd/B-NP(V), respectively) and administered to BALB/c mice intranasally. To examine the efficacy of vaccination, antibody responses, CTL responses, and morbidity/mortality after challenge were measured. Results: Both vaccines induce similar antibody and CD8 T-cell responses cross-reacting to both epitopes, and also confer cross-protection against both lineages regardless of amino acid difference. Conclusion: The rAd-vectored vaccine expressing the NP could be developed as universal influenza B vaccine which provides broader protection.
Anti-SARS-CoV-2 Nucleoprotein Antibodies Derived from Pig Serum with a Controlled Specificity
정재용,봉지홍,김홍래,박준희,이창규,강민정,김현옥,변재철 한국바이오칩학회 2021 BioChip Journal Vol.15 No.2
Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (NP) were purified from pig serum through two steps: (1) isolation of anti-NP IgG antibodies using magnetic beads with immobilized human SARS CoV-2 NP and (2) fi ltration of anti-spike protein (SP) IgG antibodies using magnetic beads with immobilized human SARS-CoV SP. The enhanced specificity of the purified antibodies to the NP of SARS-CoV-2 was demonstrated using an immunoassay with anti-NP IgG antibodies after the isolation and filtration steps. The binding constants ( K d ) of the purified anti-NP IgG antibodies to the NP of SARS-CoV-2 and the SP of SARS-CoV were estimated using a surface plasmon resonance biosensor (SPR). A competitive assay using the two-step purified anti-NP IgG antibodies from pig serum demonstrated (a) the detection of SARS-CoV-2 in viral fluid and (b) the discrimination of SARS-CoV-2 from SARS-CoV, MERS-CoV, and CoV strain 229E in viral fluids.
Satish S. Gaikwad,이현정,김지예,최강석 대한백신학회 2019 Clinical and Experimental Vaccine Research Vol.8 No.1
Purpose: The aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (DIVA) strategy accompanying the marker vaccine lacking an immunodominant epitope (IDE) of nucleoprotein of Newcastle disease virus (NDV). Materials and Methods: Recombinant epitope-repeat protein (rERP) gene encoding eight repeats of the IDE sequence (ETQFLDLMRAVANSMR) by tetra-glycine linker was synthesized. Recombinant baculovirus carrying the rERP gene was generated to express the rERP in insect cells. Specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) employing the rERP was evaluated. Results: The rERP with molecular weight of 20 kDa was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. The rERP was antigenically functional as demonstrated by Western blotting. An indirect ELISA employing the rERP was developed and its specificity and sensitivity was determined. The ELISA test allowed discrimination of NDV infected sera from epitope deletion virus vaccinated sera. Conclusion: The preliminary results represent rERP ELISA as a promising DIVA diagnostic tool.
Competitive Immunoassay of SARS‑CoV‑2 Using Pig Sera‑Derived Anti‑SARS‑CoV‑2 Antibodies
봉지홍,김태훈,정재용,이수정,성정수,이창규,강민정,김현옥,변재철 한국바이오칩학회 2021 BioChip Journal Vol.15 No.1
Anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) nucleoprotein (NP) antibodies were isolated from pig sera using human SARS-CoV-2 NP-immobilized magnetic beads. The binding properties of the isolated antibodies against SARS-CoV-2 NP were tested via flow cytometry using SARS-CoV-2 NP-immobilized magnetic beads. A competitive immunoassay was developed for detecting SARS-CoV-2 NP as well as SARS-CoV-2 in the culture fluid using magnetic beads with immobilized anti-SARS-CoV-2 NP antibodies. Selectivity tests were carried out during the competitive immunoassay for SARS-CoV, MERS-CoV, and CoV strain 229E in the culture fluid.