Wu, Jie
(Center for Disease Control and Prevention)
,
Zeng, Xian-Qiao
(Center for Disease Control and Prevention)
,
Zhang, Hong-Bin
(Gangzhou General Hospital of Guangzhou Military District)
,
Ni, Han-Zhong
(Center for Disease Control and Prevention)
,
Pei, Lei
(International Dialogues and Conflict Management)
,
Zou, Li-Rong
(Center for Disease Control and Prevention)
,
Liang, Li-Jun
(Center for Disease Control and Prevention)
,
Zhang, Xin
(Center for Disease Control and Prevention)
,
Lin, Jin-Yan
(Center for Disease Control and Prevention)
,
Ke, Chang-Wen
(Center for Disease Control and Prevention)
The highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype infect poultry and have also been spreading to humans. Although new antiviral drugs and vaccinations can be effective, rapid detection would be more efficient to control the outbreak of infections. In this study, a phage-disp...
The highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype infect poultry and have also been spreading to humans. Although new antiviral drugs and vaccinations can be effective, rapid detection would be more efficient to control the outbreak of infections. In this study, a phage-display library was applied to select antibody fragments for HPAI strain A/Hubei/1/2010. As a result, three clones were selected and sequenced. A hemagglutinin inhibition assay of the three scFvs revealed that none exhibited hemagglutination inhibition activity towards the H5N1 virus, yet they showed a higher binding affinity for several HPAI H5N1 strains compared with other influenza viruses. An ELISA confirmed that the HA protein was the target of the scFvs, and the results of a protein structure simulation showed that all the selected scFvs bound to the HA2 subunit of the HA protein. In conclusion, the three selected scFVs could be useful for developing a specific detection tool for the surveillance of HPAI epidemic strains.
The highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype infect poultry and have also been spreading to humans. Although new antiviral drugs and vaccinations can be effective, rapid detection would be more efficient to control the outbreak of infections. In this study, a phage-display library was applied to select antibody fragments for HPAI strain A/Hubei/1/2010. As a result, three clones were selected and sequenced. A hemagglutinin inhibition assay of the three scFvs revealed that none exhibited hemagglutination inhibition activity towards the H5N1 virus, yet they showed a higher binding affinity for several HPAI H5N1 strains compared with other influenza viruses. An ELISA confirmed that the HA protein was the target of the scFvs, and the results of a protein structure simulation showed that all the selected scFvs bound to the HA2 subunit of the HA protein. In conclusion, the three selected scFVs could be useful for developing a specific detection tool for the surveillance of HPAI epidemic strains.
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제안 방법
Accordingly, this study applied an scFv phage display library to select antibody fragments against the epidemic H5N1 strain A/Hubei/1/2010 isolated recently in China, for which no specific diagnostic antibodies have yet been reported. The antibodies selected via phage display screening were further evaluated for their specificities to the H5N1 virus using ELISA tests and for their binding affinity to HA using noncompetitive ELISA. Thereafter, functional studies of the selected scFvs were performed using influenza hemagglutination inhibition (HI) assays to evaluate their roles in the inhibition of hemagglutinin agglutination.
The antigen concentration, coating time, and incubation time of the antigen with the antibody were optimized by testing different antigen concentrations: 5, 2.5, 1.25, 0.625, and 0.3125 μg/ml in a 0.1 M NaHCO3 buffer (pH 8.6); different coating times: 12, 24, 36, and 48 h at 4°C in a humidity box; and different incubation times: 1, 2, 4, 8, and 16 h at room temperature.
In the third round of selection, 96 of the phage clones grown on TYE plates (15 g bacto-agar, 8 g NaCl, 10 g tryptone, 5 g yeast extract in 1 L; Sigma) were selected to be amplified and evaluated for their antigen-binding specificity using an ELISA assay. First, 96-well microtiter plates (Nunc Maxisorp plate; Gibco BRL) were coated with the inactivated H5N1 virus at a hemagglutination titer of 1:32 in a 0.
The supernatant (50 μl) of the selected phage clones was then poured into the virus-coated wells. The ELISA protocol was optimized by testing different blocking conditions using 2%, 3%, and 5% dried skimmed milk powder in PBS as the blocking buffer; different washing conditions using Tween 20 concentrations of 0.1%, 0.3%, and 0.5% in PBS as the washing buffer; and different secondary antibody concentrations of 1:1,000, 1:2,500, and 1:5,000 HRP-anti-M13 for detection. The specific affinities of the selected phage clones to the H5N1 virus were evaluated using a panel of different influenza viruses that included Enterovirus 71, Dengue virus I, pandemic H1N1 (A/Sichuan/SWL1/2009), seasonal H1N1(A/Tianjingjingnan/15/2009), seasonal H3N2 (A/Fujian tongan/196/2009), influenza B-Yamagata (B/Sichuananyue/139/2011), HPAI A/H5N1 A/Anhui/1/2005, A/Guangdong-Shenzhen/1/2011, A/Hubei/1/2010, and A/Chicken/Hong Kong/AP156/2008.
To identify the molecules with a high affinity to the inactivated H5N1 virus, three rounds of biopanning were performed to enrich the positive phage clones. The titer of the phage eluate from the first round (2.
The amplified fragments for three unique scFv molecules, named A1 (A1, C3, and C12 are the same), A10 (A10, D4, and H7 are the same), and F11, matched the expected scFv fragments, and these three scFV molecules were then sequenced. In all three scFvs, mutated sites were seen at positions 50–53 and 91–96 of the kappa chain and positions 50–58 and 95–98 of the heavy chain.
3. Specific affinity of the three selected phage clones to the H5N1 virus, detected using an ELISA to study their binding activity with 10 inactivated viruses.
대상 데이터
Using sequential biopanning to screen a specific phage display library (Human Single Fold Tomlison I+J scFv Libraries), this study identified three unique scFvs that exhibited a specific binding affinity to several strains of HPAI A/H5N1, including A/Hubei/1/2010, A/Guangdong-Shenzhen/1/2011, A/Chicken/HongKong/AP156/2008, and A/Anhui/1/2005, representing the main strains causing H5N1 infection in China. All three fragments included mutations at similar positions, indicating that they probably bind to very similar epitopes and are highly specific to HPAI A/H5N1 strains [9, 26].
이론/모형
The HA sequence for Influenza B and A/Chicken/Hong Kong/AP156/2008(H5N1) were not included in this alignment owing to a significant sequence discrepancy and unavailability in the online database, respectively. All the downloaded amino acid sequences were subjected to MEGA5 and the alignments conducted using MUSCLE methods.
3, including HPAI H5N1 strains A/Hubei/1/2010, A/Anhui/1/2005, A/Guangdong-Shenzhen/1/2011, pandemic H1N1 A/Sichuan/SWL1/2009, seasonal H1N1 A/Tianjingjingnan/15/2009, and seasonal H3N2 A/Fujian tongan/196/2009, were all downloaded from the flu sequence database of the China CDC. The alignments were accomplished in MEGA5 using MUSCLE methods.
후속연구
Hence, antibodies highly specific to H5 subclade strains are ideal candidates for the development of new diagnostic kits. Moreover, the selected molecules specific to the the HA2 subunit of the H5N1 virus identified in this study can be further developed for use in clinical diagnostic tests for humans and for surveillance of H5N1 infections in birds and the external environment.
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