Epitope-Containing Short Peptides Capture Distinct IgG Serodynamics That Enable Differentiating Infected from Vaccinated Animals for Live-Attenuated Vaccines

Figures

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  FIG 1. Screening for ECSPs that possess both unique IgG serodynamics and diagnostic potential. (A) Longitudinal samples were prepared through immunization of goats with a live-attenuated vaccine, and blood samples were collected at specific intervals. Latitudinal samples included vaccinated sera and negative sera from naive goats without vaccination or infection. (B) Overview of screening with the peptide microarray platform; it is a modified indirect ELISA configured with chemiluminescence, and the whole test takes ∼1 h. (C) Workflow of cELISA (∼1.5 h) and (D) VNT (∼7 days). (E, Left) Schematic of microarray 1. It constitutes four subarrays; each subarray contains four positive control points (red, goat IgG), one negative control point (yellow, printing buffer), and 47, 31, 49, and 56 20-mer peptides (blue) from the N, M, F, and H proteins of PPRV, respectively. The missing blue dots within each subarray indicate that the corresponding peptides were not able to be synthesized. (E, Middle and Right) Two representative chemiluminescence images from the peptide microarray with longitudinal samples at 0 dpv (middle) and 18 dpv (right) from the T3-2 goat.

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  FIG 2. Distinct serodynamics and epitope type changes of IgGs. (A) Sixty-four longitudinal sera collected from a representative goat (T3-2). (B) The clustered heat map of the 64 longitudinal sera from T3-2 against 183 peptides (microarray 1). (C) Comparison of the IgG serodynamics from 64 longitudinal sera of T3-2 detected by ECSPs (N49 and F10), the cELISA kit, and VNT, respectively. (D) The layout of microarray 2. PC is the goat IgG, and NC is the printing buffer; these are the positive and negative controls, respectively, for horseradish peroxidase (HRP)-conjugated antibody interaction. (E) Results of serum T3-2 (169 dpv) and (F) T3-2 (18 dpv) incubated with microarray 2 after different denaturing treatments. (G) Blocking experiment with N protein incubated with T3-2 (18 dpv). Treatment with 20 mM DTT alone showed no signal change on any of the ECSPs and proteins (data not shown).

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  FIG 3. Validation of distinct IgG serodynamics, using latitudinal sera, which have implications for vaccine titer evaluation and DIVA. For individual ECSPs or proteins of microarray 2, the result was positive if its S/P was larger than the cutoff. For the ECSP combination (i.e., N47 + N49 + N50 + F10), the result was positive if any ECSP was positive. For microarray 2, the result was positive if any two proteins were positive (mainly for titer evaluation) or if any ECSP was positive (mainly for DIVA). (A) Selected ECSPs and proteins. (B) Six groups of sera (C1 through C6) were collected to test the microarray 2. The positive/negative status of these latitudinal samples were confirmed by both cELISA and VNT. (C) Representative images of negative and positive serum. Results of sera against microarray 2 are as follows: (D) C1, the 267 negative sera. (E, Left) One hundred thirty positive sera (C2 and C3 combined). (E, Middle) C4, 8 positive sera at 480 dpv. (E, Right) C5, 17 positive sera at 930 dpv. (F) C6, 25 positive sera due to virus infection.

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  FIG 4. Denaturation and blocking experiments. (A) Flowchart of denaturation experiments (see Fig. 2E and F for data). (B) Three groups of anti-F IgGs. (C) Flowchart of blocking experiments. Free ECSP or protein was individually doped to serum before screening against microarray 2. (D) Results of blocking experiments.

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  FIG 5. A general protocol for multiple purposes: vaccine titer evaluation, DIVA diagnosis, and surveillance as suitable application scenarios. (A) Proposed serum sampling schedule. (B) Serum sampling points matched with IgG serodynamics. (C) With use of the PPR live-attenuated vaccine to mimic the virus infection, the spiked ECSPs response clearly indicated an infection.