Vaccination with recombinant oncosphere antigens reduces the susceptibility of sheep to infection with Taenia multiceps

Abstract

Taenia multiceps is a cestode parasite, the larval stage of which encysts in the brain of sheep, goats and cattle causing an often fatal condition. The parasite also causes zoonotic infections in humans. Homologues of the recombinant oncosphere vaccine antigens from Taenia ovis and other Taenia species were identified in T. multiceps. Sequencing of the associated T. multiceps genes and cloning of the encoding mRNA has revealed conserved features in the genes and proteins. The T. multiceps oncosphere proteins, designated Tm16 and Tm18, contain a predicted secretory signal and fibronectin type III domain. The recombinant Tm16 and Tm18 proteins were successfully expressed in Escherichia coli as fusion proteins with GST. The antigens, formulated with Quil A adjuvant, were tested in a vaccine trial in sheep. The antigens stimulated immunity in sheep against challenge infection with T. multiceps eggs. Five of nine control sheep died due to a challenge infection with T. multiceps whereas none of 20 vaccinated animals died as a result of the parasite challenge (P=0.001). In addition, vaccination with the Tm16 protein, or Tm16 plus Tm18, induced significant protection against the number of parasites encysting in the brain as a result of the challenge infection (P=0.023, P=0.015, respectively). No clear relationship was apparent between the level of specific serum antibody in vaccinated animals and either the presence or absence of parasites or the number of parasites that occurred in some of the vaccinated animals. We believe this study is the first description of recombinant vaccine-related investigations for T. multiceps. The recombinant oncosphere antigens identified may allow development of effective vaccination strategies against T. multiceps infection in sheep. They raise the potential for the development of a combined vaccine with the Echinococcus granulosus EG95 antigen for prevention of T. multiceps as well as preventing the transmission of cystic hydatid disease.

Fig. 1

Diagrammatic representation of the gene structure of tm16 and tm18. Each gene has a conserved structure that consists of three exons (represented by black boxes labelled e1–e3) and two introns (lines) in the protein-encoding region. Grey lines represent introns in the 3′ untranslated region (UTR) and grey bars show 3′ UTR regions that have been identified in the cloned mRNA of each gene. Numbers below each gene represent the length, in bp, of each intron or exon.

Fig. 2

Alignment of the amino acid sequence predicted from cloned mRNA of tm16 (A) and tm18 (B) from T. multiceps with oncosphere antigens from other Taenia species. Amino acid mismatches are shaded. Boxed amino acids (1–16 for Tm16, A, and 1–18 for Tm18, B) encode putative secretory signals. Horizontal lines below the alignments define the polypeptide regions encoded by the three exons (e1–e3). Key amino acid residues defining the fibronectin type III domain are highlighted with arrows. GenBank Accession Nos. are as follows; To16 (U89944), TSOL16 (AY147841), To18 (U89943), TSOL18 (AF017788) and TSA18 (X98577).

Fig. 3

SDS–PAGE of soluble and insoluble protein extracts of Escherichia coli cultures expressing GST fusion proteins and affinity purified products. (A) Soluble fraction (lane1), insoluble fraction (lane 2) and purified Tm16-GST (lane 3) from E. coli culture expressing Tm16. (B) Soluble fraction (lane1), insoluble fraction (lane 2) and purified Tm18-GST (lane 3) from E. coli culture expressing Tm18. Molecular weight markers (kDa) are shown on the left of each panel.

Fig. 4

Sheep brain following experimental infection with Taenia multiceps and a coenurus removed from the left parieto-occipital region. The scheme used for notation of the location of coenuri in the brains of infected sheep is shown on the right-hand side (after Edwards and Herbert, 1982).

Fig. 5

Specific serum antibody responses detected in ELISA to Tm16 and Tm18 antigens in sheep receiving three immunisations (V1–V3) with 50 μg of either antigen alone (A and B) or receiving 50 μg of each protein (C and D) plus 1 mg Quil A adjuvant. All animals were challenged with T. multiceps 2 weeks after the third injection. Different symbols represent serum antibody responses in individual animals.

Fig. 6

Specific serum antibody responses detected in ELISA to the recombinant antigens (ordinate) and GST (abscissa) in sheep vaccinated with the Tm16 (A) and Tm18 (B) antigens.

Fig. 7

Relationship between the titre of specific antibody against Tm16 or Tm18 antigens detected in individual animals at the time of challenge infection and the presence (solid symbols) or absence (open circles) of parasites detected at necropsy. Groups of sheep were vaccinated either with the Tm16 or Tm18 antigen alone or with a combination of both Tm16 and Tm18. A and B represent total-specific IgG to Tm16 and Tm18, respectively.