Identification of protective antigens following HES immunization.

<p><b>A-B</b>. Day 28 post-challenge worm and fecal egg burdens (d 14, 21, 28) in C57BL/6 mice immunized with native or heat-treated HES. Data pooled from two experiments. Significance determined by ANOVA as indicated. <b>C</b>. Protective antibody targets revealed by immunoprecipitation of biotin-labeled HES by serum antibodies from immunized mice. Immunoprecipitated proteins were separated by pI (range 3–10) and molecular weights (indicated in kDa) and visualized with streptavidin HRP. Blue, red, green and brown circles represent VAL-1, 2, 3 and 4, respectively. Unknown antigens circled black. Sera pooled from 5 HES-vaccinated C57BL/6 mice pre-challenge and representative of two independent experiments. <b>D</b>. Day 28 post-challenge worm burdens from C57BL/6 mice immunized with a combination of VAL-1,-2 and -3, or with HES depleted of these 3 antigens. Data pooled from two experiments. Significance determined by ANOVA <i>Vs</i> PBS/alum. <b>E-F</b>. Immunoprecipitation of biotin-labeled HES antigens with vaccination sera as (C) from mice immunized with VAL-1/2/3 (E), or VAL-1/2/3 depleted HES (F). <b>G</b>. LC-MS/MS identification of antibody targets in mice immunized with VAL-1/2/3-depleted HES, performed on samples of unlabeled HES immunoprecipitated with serum antibodies from these mice. Proteins ranked according to spectral count (“count”). “Unique” represents number of unique peptide sequences in identification, “score” is Mascot score, and emPAI estimated abundance also shown. Highest scoring BLAST homolog with expect values indicated. <b>H</b>. Day 28 post-challenge worm burdens from C57BL/6 mice immunized with HES or HES depleted of VAL-1, -2, -3 and -4. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004676#ppat.1004676.s005" target="_blank">S5 Fig</a>.