In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16

Figures

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  Figure 1. Immune response analysis before each immunization. (A) Simplified diagram of vaccination protocol with primary injection, then first booster through booster number 8. (B) The binding ability of purified immunoglobulin yolk (IgY)-binding protein antibodies against CA16-infected lysate and bovine serum albumin (BSA). The abbreviations pre- or 8th-vac, etc., represent pre-vaccination through to the eighth vaccination on a weekly basis. The numbers 1, 2, or 3 mean the number of eggs IgY purified at that vaccination. The ELISA assay was run to confirm the immune response induction in the laying hen. Zika virus envelope (ZKV_E) IgY 7th-vac was used as a negative antibody control. (C) An ELISA test was conducted to reveal the total IgY antibody at dilutions of 1/500 to 1/1,024,000. The assay compared the pre-vaccination IgY titer to the 8th-vaccination IgY titer. The experiments were run in duplicate and data presented as average ± standard deviation (SD).

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  Figure 2. Characterization of immunoglobulin yolk-binding protein by Western blot. SDS-PAGE displaying CA16-infected lysate (lane 1), non-infected (RD cells) lysate (lane 2), and BSA (lane 3) stained with Coomassie blue dye; lane M: protein marker (A). The samples were transferred onto PVDF membranes and detected with pre-vaccination IgY (B); 1st-vaccination IgY (C); 3rd-vaccination IgY (D); 5th-vaccination IgY (E); 6th-vaccination IgY (F); 7th-vaccination IgY (G); and 8th-vaccination IgY (H) antibodies at a dilution of 1:3000. The complex was then revealed by HRP-conjugated donkey anti-chicken IgY antibodies at a dilution of 1:10,000. BSA and non-infected lysate were used as negative antigen controls.

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  Figure 3. Schematic overview of single-chain variable fragment (scFv) gene cloning into the pComb3X plasmid. The arrangement of the VH and VL regions joined by a flexible linker representing the anti-CA16 scFv gene (A). The pComb3X gene constructs contained histidine (His6×) tag and hemagglutinin (HA) tag for detection and purification of recombinant scFv antibodies (B). The g3p fragment was included for the phage to display the scFv antibody protein on the surface of the viral particles.

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  Figure 4. Construction analysis of the scFv-binding protein antibodies phage display libraries and insert confirmation. PCR products of lane 1: VH-S (variable region of heavy chain with short-linker); lane 2: VH-L (variable region of heavy chain with long-linker); and lane 3: VL (variable region of light chain) (A). PCR products of the scFv-S (lane 1) and scFv-L (lane 2) (B). PCR amplification of full-length scFv genes from the phage display library for confirming insertion frequency (C). Lanes 1–8: scFv genes containing VH-peptide linker-VL fragments with short- or long-linkers, respectively. All three panels have DNA ladder (lane M) as a reference point.

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  Figure 5. The phage titers, colony-PCR, scFv selection, and their specific binding abilities. (A) The recombinant eluted phage titer was checked after the first cycle of panning and throughout the five cycles. Their eluted titer was demonstrated using a colony formation assay. (B) After each cycle of the bio-panning process, colonies were randomly selected to confirm the phagemid DNA of the binders; DNA ladder (M) was used for a reference point. (C) Amplified phage-binders from each cycle of bio-panning were tested for their binding activities. These eluted titers and their abilities to bind were compared with phages from original libraries (0). (D) The ELISA result showed the binding ability of individual clones with short-linkers. IgY-binding protein antibody (1:3000 dilution) was included as a positive control. HRP-conjugated rabbit anti-mouse IgG and HRP-conjugated donkey anti-chicken IgY antibodies were used to confirm the complex at 1:10,000 dilutions, respectively.

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  Figure 6. Expression and grouping analysis of scFv antibody. (A) Thirteen randomly selected scFv clones were loaded onto respective wells of SDS-PAGE. (B) The PVDF membrane was probed by mouse anti-HA tag antibodies at 1:5000 dilutions, then detected by HRP-conjugated rabbit anti-mouse IgG antibodies at 1:5000 dilutions. The expected scFv molecular mass was about 26 kDa. (C) All the scFv antibodies had the same amino acid sequences and were classified as one group.

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  Figure 7. Analysis of IgY and scFv neutralizing activity against coxsackievirus A16. (A) Pre-vaccination IgY as negative control antibody with an IC50 greater than 570 µg/mL. (B) Immunoglobulin yolk (8th vaccination) antibody with an IC50 equal to 0.67 ± 0.06 µg/mL. (C) Scheme 50. equal to 61.46 ± 1.75 µg/mL. All experiments were carried out in triplicate. The results are presented as means plus or minus standard deviation.

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  Figure 8. Cross-reactive analysis of scFv and IgY antibodies against coxsackievirus A16 and enterovirus 71. (A) SDS-PAGE showing protein marker (lane M); CA16-infected lysate (lane 1); EV71-infected lysate (lane 2); non-infected lysate (lane 3); BSA protein (lane 4). Western blot assay probed with (B) scFv-binding protein S1 and (C) IgY antibodies. (D) The ELISA test was carried out by the addition of scFv antibody and mouse anti-HA tag, followed by HRP-conjugated rabbit anti-mouse IgG antibodies. The BSA was included as an antigen control on both the Western blot and ELISA assays.