Characterization of S3Pvac anti-cysticercosis vaccine components: implications for the development of an anti-cestodiasis vaccine

Abstract

Background: Cysticercosis and hydatidosis seriously affect human health and are responsible for considerable economic loss in animal husbandry in non-developed and developed countries. S3Pvac and EG95 are the only field trial-tested vaccine candidates against cysticercosis and hydatidosis, respectively. S3Pvac is composed of three peptides (KETc1, GK1 and KETc12), originally identified in a Taenia crassiceps cDNA library. S3Pvac synthetically and recombinantly expressed is effective against experimentally and naturally acquired cysticercosis.

Methodology/principal findings: In this study, the homologous sequences of two of the S3Pvac peptides, GK1 and KETc1, were identified and further characterized in Taenia crassiceps WFU, Taenia solium, Taenia saginata, Echinococcus granulosus and Echinococcus multilocularis. Comparisons of the nucleotide and amino acid sequences coding for KETc1 and GK1 revealed significant homologies in these species. The predicted secondary structure of GK1 is almost identical between the species, while some differences were observed in the C terminal region of KETc1 according to 3D modeling. A KETc1 variant with a deletion of three C-terminal amino acids protected to the same extent against experimental murine cysticercosis as the entire peptide. On the contrary, immunization with the truncated GK1 failed to induce protection. Immunolocalization studies revealed the non stage-specificity of the two S3Pvac epitopes and their persistence in the larval tegument of all species and in Taenia adult tapeworms.

Conclusions/significance: These results indicate that GK1 and KETc1 may be considered candidates to be included in the formulation of a multivalent and multistage vaccine against these cestodiases because of their enhancing effects on other available vaccine candidates.

Figure 1. Multiple sequence alignment of KETc1 peptides.

The DNA and amino acid sequences of KETc1 of several species of the Taeniidae family were aligned. KETc1 alignments of amino acids (A) and cDNA (B) are shown in the upper part of the figure. On every alignment, boxes corresponding to the regions subjected to dN/dS-ratio analysis are marked accordingly. Panel C and D shows the 3D models of the complete and truncated version of KETc1. Panel D depicts the models corresponding to T. solium (red), T. crassiceps ORF and WFU (pink), T. saginata (light green) and Echinococcus group (dark green). Structures are aligned with very good general agreement. Panel D shows the alignment of the truncated KETc1 peptide where the yellow model corresponds to T. solium and T. saginata; blue corresponds to T. crassiceps ORF and WFU and brown to the Echinococcus group.

Figure 2. Multiple sequence alignment GK1 peptides.

The DNA and amino acid sequences of GK1 in several species of the Taeniidae family were aligned. GK1 alignments of amino acids (A) and cDNA (B) are shown in the upper part of the figure; the region encoding the actual peptides is marked with a double-headed arrow on the top of the alignment. On every alignment, boxes corresponding to the regions subjected to dN/dS-ratio analysis are marked accordingly. Models of the 3D structure of the GK1 peptide are shown in panel C. The model in red corresponds to T. solium, T. saginata and E. granulosus' GK1 and in purple to T. crassiceps ORF and WFU and E. multilocularis. A very good fitting between the two structures is shown as expected from the strong similarity of the amino acid sequence.

Figure 3. Immunolocalization of the S3Pvac peptides in larval specimens of the four Taeniidae families.

Metacestode cryostat parasite sections were incubated with a rabbit pre-immune serum (A) or with the three different rabbit antibodies (anti-GK1, anti-KETc1, anti-KETc12) and developed with biotinylated goat anti-rabbit IgG plus streptavidin peroxidase conjugate and counterstained with hematoxilin. The arrows indicate the areas in the metacestode where the peptides were localized. 10X (A and C), 40X (B and D). Tegument (T), parenchyma (P), tegument of the spiral canal (TSC), parenchymal folds (PF), microthrix (MT), parenchyma (P). The three peptides are present in metacestode stage of four cestodes analyzed.

Figure 4. S3Pvac peptides were detected in the tegument of Taenia tapeworms and T. solium eggs.

No reaction was detected in control sections, for which naive rabbit serum was used in place of the specific polyclonal rabbit serum. S.- Sucker; Sc. Scolex, MP.- Medullar parenchyma, N.- Neck; DC.- Distal cytoplasm region; PE.- perinuclear cytoplasm region. O.- oncospheres.

Figure 5. Immunolocalization of the S3Pvac peptides in E. granulosus and E. multilocularis.

Sections of larval specimens reveal a strong binding to the tegument (T) as well as in the parenchyma (P) of E. multilocularis and in tegument of E. granulosus. (A) Control labeled with rabbit pre immune serum. (B) Positive control labeled with polyclonal sera from rabbit immunized with a total extract of Taenia crassiceps ORF cysticerci.