• Journal of Veterinary Science
• /
• v.23 no.2
• /
• pp.24.1-24.10
• /
• 2022

Background: Small interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia. Objectives: The main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus. Methods: The effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells. Results: The siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene. Conclusions: These findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.10
• /
• pp.1683-1690
• /
• 2018

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.

• IMMUNE NETWORK
• /
• v.9 no.5
• /
• pp.169-178
• /
• 2009

DNA immunization induces B and T cell responses to various pathogens and tumors. However, these responses are known to be relatively weak and often transient. Thus, novel strategies are necessary for enhancing immune responses induced by DNA immunization. Here, we demonstrated that co-immunization of influenza virus nucleoprotein (NP) gene significantly enhances humoral and cell-mediated responses to codelivered antigens in mice. We also found that NP DNA coimmunization augments in vivo proliferation of adoptively transferred antigen-specific CD4 and CD8 T cells, which enhanced protective immunity against tumor challenge. Our results suggest that NP DNA can serve as a novel genetic adjuvant in cocktail DNA vaccination.

• IMMUNE NETWORK
• /
• v.23 no.4
• /
• pp.32.1-32.15
• /
• 2023

Most influenza vaccines currently in use target the highly variable hemagglutinin protein to induce neutralizing antibodies and therefore require yearly reformulation. T cell-based universal influenza vaccines focus on eliciting broadly cross-reactive T-cell responses, especially the tissue-resident memory T cell (T_RM) population in the respiratory tract, providing superior protection to circulating memory T cells. This study demonstrated that intramuscular (i.m.) administration of the adenovirus-based vaccine expressing influenza virus nucleoprotein (rAd/NP) elicited weak CD8 T_RM responses in the lungs and airways, and yielded poor protection against lethal influenza virus challenge. However, a novel "prime-and-deploy" strategy that combines i.m. vaccination of rAd/NP with subsequent intranasal administration of an empty adenovector induced strong NP-specific CD8⁺ T_RM cells and provided complete protection against influenza virus challenge. Overall, our results demonstrate that this "prime-and-deploy" vaccination strategy is potentially applicable to the development of universal influenza vaccines.

• IMMUNE NETWORK
• /
• v.21 no.4
• /
• pp.28.1-28.14
• /
• 2021

Lung-resident memory T cells (T_RM) play an essential role in protecting against pulmonary virus infection. Parenteral administration of DNA vaccine is generally not sufficient to induce lung CD8 T_RM cells. This study investigates whether intramuscularly administered DNA vaccine expressing the nucleoprotein (NP) induces lung T_RM cells and protects against the influenza B virus. The results show that DNA vaccination poorly generates lung T_RM cells and massive secondary effector CD8 T cells entering the lungs after challenge infection do not offer sufficient protection. Nonetheless, intranasal administration of non-replicating adenovirus vector expressing no Ag following priming DNA vaccination deploys NP-specific CD8 T_RM cells in the lungs, which subsequently offers complete protection. This novel 'prime and deploy' strategy could be a promising regimen for a universal influenza vaccine targeting the conserved NP Ag.

• Journal of Life Science
• /
• v.30 no.9
• /
• pp.749-762
• /
• 2020

Influenza A viruses are circulating in a variety of hosts, including humans, pigs, and poultry. Swine influenza virus is a zoonotic pathogen that can be readily transmitted to humans. The influenza viruses of the 2009 H1N1 pandemic were derived from swine influenza viruses, and it has been suggested that the 1957 H2N2 pandemic and the 1968 H3N2 pandemic both originated in pigs. Pigs are regarded as a mixing vessel in the creation of novel influenza viruses since they are readily infected with human and avian influenza viruses. We isolated three novel H1N2 influenza viruses from pigs showing respiratory symptoms on a Korean farm in 2019. These viruses were reassortants, containing PA and NP genes from those of the 2009 H1N1 influenza virus in addition to PB2, PB1, HA, NA, M, and NS genes from those of triple-reassortant swine H3N2 and classical swine H1N2 influenza viruses circulating in Korean pigs. Mice infected with the isolated H1N2 influenza virus lost up to 17% body weight and exhibited interstitial pneumonia involving infiltration of many inflammatory cells. Results suggest that close surveillance to detect emerging influenza viruses in pigs is necessary for the health of both pigs and humans.

• Korean Journal of Clinical Laboratory Science
• /
• v.42 no.1
• /
• pp.1-15
• /
• 2010

Swine influenza virus (SIV) or swine-origin influenza virus (S-OIV) is endemic in swine, and classified into influenza A and influenza C but not influenza B. Swine influenza A includes H1N1, H1N2, H3N1, H3N2 and H2N3 subtypes. Infection of SIV occurs in only swine and that of S-OIV is rare in human. What human can be infected with S-OIV is called as zoonotic swine flu. Pandemic 2009 swine influenza H1N1 virus (2009 H1N1) was emerged in Mexico, America and Canada and spread worldwide. The triple-reassortant H1N1 resulting from antigenic drift was contained with HA, NA and PB1 of human or swine influenza virus, PB2 and PA polymerase of avian influenza virus, and M, NP and NS of swine influenza virus, The 2009 H1N1 enables to transmit to human and swine. The symptoms and signs in human infected with 2009 H1N1 virus are fever, cough and sore throat, pneumonia as well as diarrhea and vomiting. Co-infection with other viruses and bacteria such as Streptococcus pneumoniae can occur high mortality in high-risk population. 2009 H1N1 virus was easily differentiated from seasonal flu by real time RT-PCR which contributed rapid and confirmed diagnosis. The 2009 H1N1 virus was treated with NA inhibitors such as oseltamivir (Tamiflu) and zanamivir (Relenza) but not with adamantanes such as amantadine and rimantadine. Evolution of influenza virus has continued in various hosts. Development of a more effective vaccine against influenza prototypes is needed to protect new influenza infection such as H5 and H7 subtypes to infect to multi-organ and cause high pathogenicity.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.5
• /
• pp.809-815
• /
• 2018

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.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.2
• /
• pp.304-316
• /
• 2021

Vaccination is the most effective way to prevent influenza virus infections. However, conventional vaccines based on hemagglutinin (HA) have to be annually updated because the HA of influenza viruses constantly mutates. In this study, we produced a 3M2e-3HA2-NP chimeric protein as a vaccine antigen candidate using an Escherichia coli expression system. The vaccination of chimeric protein (15 ㎍) conferred complete protection against A/Puerto Rico/8/1934 (H1N1; PR8) in mice. It strongly induced influenza virus-specific antibody responses, cytotoxic T lymphocyte activity, and antibody-dependent cellular cytotoxicity. To spare the dose and enhance the cross-reactivity of the chimeric, we used a complex of poly-γ-glutamic acid and alum (PGA/alum) as an adjuvant. PGA/alum-adjuvanted, low-dose chimeric protein (1 or 5 ㎍) exhibited higher cross-protective effects against influenza A viruses (PR8, CA04, and H3N2) compared with those of chimeric alone or alum-adjuvanted proteins in vaccinated mice. Moreover, the depletion of CD4+ T, CD8+ T, and NK cells reduced the survival rate and efficacy of the PGA/alum-adjuvanted chimeric protein. Collectively, the vaccination of PGA/alum-adjuvanted chimeric protein induced strong protection efficacy against homologous and heterologous influenza viruses in mice, which suggests that it may be a promising universal influenza vaccine candidate.

• Korean Journal of Veterinary Service
• /
• v.40 no.3
• /
• pp.187-192
• /
• 2017

In this report, fifteen monoclonal antibodies (MAbs) against an avian influenza virus (H9N2 subtype) were newly produced and characterized. These MAbs proved to react to the epitopes of nucleocapsid protein (NP), hemagglutinin (HA), neuraminidase (NA) and non-structural protein 1 (NS1) of Korean H9N2 strain, respectively. Two HA-specific MAbs showed the ability to inhibit the hemagglutination activity of H9N2 subtype avian influenza virus when tested by hemagglutination inhibition (HI) assay. All MAbs did not cross-react with other avian-origin viruses (Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus and avian rotavirus) by immunofluorescence test or enzyme-linked immunosorbent assay. The MAbs produced in this study could be useful as the materials for diagnostics and therapeutics against Korean-lineage H9N2 virus infections.