Epitope Mapping of Exposed Tegument and Alimentary Tract Proteins Identifies Putative Antigenic Targets of the Attenuated Schistosome Vaccine

Abstract

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

Keywords: Schistosoma mansoni; alimentary tract; antigenic targets; attenuated vaccine; epitope mapping; tegument proteins.

Figure 1

Diagram showing tissues where proteins printed on the array originate and the list of proteins in each category. These tissues represent the major parasite-host interface where antigens are exposed or released into the blood stream.

Figure 2

Properties of sera from IFNgR KO mouse used to screen the arrays. (A) Percent protection of C57Bl/6 versus IFNgR KO mice given one, two or three vaccinations with 500 radiation-attenuated cercariae and then challenged 5 weeks later with 200 normal cercariae. (B) Antigen specific IgG1 antibody levels in sera recovered from these mice 14 days after each vaccination. (C) Passive protection against a 200 cercarial percutaneous challenge of naïve C57Bl/6 recipients given injections of serum recovered from 3x vaccinated C57Bl/6 or IFNgR KO mice on the designated days. MT2 and MT3 sera were used to screen the four peptide arrays.

Figure 3

Heatmaps of the mean reactivities of murine serum pools against the peptide arrays: 1) short alimentary tract; 2) longer alimentary tract; 3) short tegument surface; 4) long tegument surface. V, mean of three C57Bl/6 samples from mice vaccinated with three exposures of 500 radiation-attenuated cercariae. G, mean of two IFNgR KO samples, MT2 and MT3 passive transfer experiments. I, mean of three C57Bl/6 samples from mice exposed to 500 normal cercariae for 4 weeks. $\overline{\mathrm{x}}$, mean of all eight pools.

Figure 4

Bar chart summarizing the reactivity of all proteins on the four arrays, based on the data in Supplementary Table 2. The y axis is cumulative Agilent peptide score above zero, ignoring protein length for each protein. The means of three I, three V, and two G results are plotted plus one control serum. Note the much weaker response of tegument compared to alimentary tract proteins (y axis range).

Figure 5

The mean reactivity of array proteins based on the data in Supplementary Table 2, normalized for number of amino acids and segregated into four groups by tissue of origin. The differences in group reactivity were tested for significance using a t-test. Note the log y axis scale.

Figure 6

Reactive regions mapped onto six selected crystal structures using CCP4mg (50). (A) Aspariginyl endopeptidase. (B) Cathepsin B1. (C) Apoferritin. (D) Calpain. (E) Carbonic anhydrase. (F) VAL-7. The protein chain is colored ice-blue in ribbon format. The position of each reactive region listed in Supplementary Table 3 is shown in worm format and selected key peptides from Table 1 or Supplementary Table 3 are indicated by P number. The worms are colored by solvent accessibility ranging from buried (blue) to accessible (red) on a scale 0-200 Ų, based on the algorithm described by Lee and Richards (51); the detailed key for each model is illustrated in Supplementary Figure 4. Where present, inhibitors/substrates are displayed in the active site of enzymes in ball and stick format, with atoms colored using a modified Corey/Pauling/Koltan (CPK) convention, to indicate their location, while the side chains of amino acids comprising the active site or other named features are highlighted as cylinders.