Preparation of monoclonal antibodies against poor immunogenic avian influenza virus proteins

He, Jijun; Hsieh, Ming-Shou; Chiu, Yi-Chung; Juang, Rong-Huay; Wang, Ching-Ho

doi:10.1016/j.jim.2012.09.009

Cited by 13 publications

(8 citation statements)

References 48 publications

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“…Many epitopes have been defined and mapped in the NS1 protein of influenza A majority subtypes. The following epitopes were previously mapped and included part of the epitope identified in this study: CFLWHVRKRVADQELGDAPF [ 31 ], ELGDAPFLDRLRRDQ, RVADQELGDAPFLDR [ 31 ], ADQELGDAPFLDRLRRD-QKS [ 32 ], DAPFLDRL, QELSDAPFL, DQDLGDAPFLDRLRRDQ, DQELSDAPF, RKQVADQDLGDAPFLDR [ 33 , 34 ], GDAPFLDRLR and GDAPFLDRLRRDQKS-LRGRG [ 35 ]. These epitopes contained 8 to 20 amino acids, and all contained the DAPF motif.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

et al. 2016

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Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.

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Section: Discussionmentioning

confidence: 99%

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

et al. 2016

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“…There are also three epitopes that contain part of the epitope identified in this study. They are VKNAIGVLIGGLEWNDN [ 29 ], DEDVKNAIGVLIGGLEWNDN [ 33 ], and NAVGVLIGGLEWNDN [ 30 ]. Because none of these epitopes are completely identical to the epitope described in this study, this new epitope is another different linear epitope on the AIV NS1 protein.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel linear epitope on the NS1 protein of avian influenza virus

et al. 2015

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BackgroundThe NS1 protein of avian influenza virus (AIV) is an important virulent factor of AIV. It has been shown to counteract host type I interferon response, to mediate host cell apoptosis, and to regulate the process of protein synthesis. The identification of AIV epitopes on NS1 protein is important for understanding influenza virus pathogenesis.ResultsIn this paper, we describe the generation, identification, and epitope mapping of a NS1 protein-specific monoclonal antibody (MAb) D9. First, to induce the production of MAbs, BALB/c mice were immunized with a purified recombinant NS1 expressed in E. coli. The spleen cells from the immunized mice were fused with myeloma cells SP2/0, and through screening via indirect ELISAs, a MAb, named D9, was identified. Western blot assay results showed that MAb D9 reacted strongly with the recombinant NS1. Confocal laser scanning microscopy showed that this MAb also reacts with NS1 expressed in 293T cells that had been transfected with eukaryotic recombinant plasmids. Results from screening a phage display random 7-mer peptide library with MAb D9 demonstrated that it recognizes phages displaying peptides with the consensus peptide WNLNTV--VS, which closely matches the 182WNDNTVRVS190 of AIV NS1. Further identification of the displayed epitope was performed with a set of truncated polypeptides expressed as glutathione S-transferase fusion proteins, and the motif 182WNDNT186 was defined as the minimal unit of the linear B cell epitope recognized by MAb D9 in western blot assays. Moreover, homology analysis showed that this epitope is a conserved motif among AIV.ConclusionsWe identified a conserved linear epitope, WNDNT, on the AIV NS1 protein that is recognized by MAb D9. This MAb and its epitope may facilitate future studies on NS1 function and aid the development of new diagnostic methods for AIV detection.

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“…The monoclonal antibody (mAb) D9 was able to interact with an avian influenza virus' epitope of NS1, although not inhibiting it [131]. Still, regarding avian influenza, several other mAbs were found to bind to virus' proteins, in particular, NS1 [132]. In a different study, mAb 2H6 was found to bind to NS1, inhibiting the interaction between NS1 and double-stranded RNA [112].…”

Section: Experimental and In Silico Approaches Towards A New Therapeumentioning

confidence: 96%

The Central Role of Non-Structural Protein 1 (NS1) in Influenza Biology and Infection

Rosário‐Ferreira

Preto

Melo

et al. 2020

IJMS

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Influenza (flu) is a contagious viral disease, which targets the human respiratory tract and spreads throughout the world each year. Every year, influenza infects around 10% of the world population and between 290,000 and 650,000 people die from it according to the World Health Organization (WHO). Influenza viruses belong to the Orthomyxoviridae family and have a negative sense eight-segment single-stranded RNA genome that encodes 11 different proteins. The only control over influenza seasonal epidemic outbreaks around the world are vaccines, annually updated according to viral strains in circulation, but, because of high rates of mutation and recurrent genetic assortment, new viral strains of influenza are constantly emerging, increasing the likelihood of pandemics. Vaccination effectiveness is limited, calling for new preventive and therapeutic approaches and a better understanding of the virus–host interactions. In particular, grasping the role of influenza non-structural protein 1 (NS1) and related known interactions in the host cell is pivotal to better understand the mechanisms of virus infection and replication, and thus propose more effective antiviral approaches. In this review, we assess the structure of NS1, its dynamics, and multiple functions and interactions, to highlight the central role of this protein in viral biology and its potential use as an effective therapeutic target to tackle seasonal and pandemic influenza.

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Preparation of monoclonal antibodies against poor immunogenic avian influenza virus proteins

Cited by 13 publications

References 48 publications

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Identification of a novel linear epitope on the NS1 protein of avian influenza virus

The Central Role of Non-Structural Protein 1 (NS1) in Influenza Biology and Infection

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