A Pilot Study on Developing Mucosal Vaccine against Alveolar Echinococcosis (AE) Using Recombinant Tetraspanin 3: Vaccine Efficacy and Immunology

Humans and rodents become infected with E. multilocularis by oral ingesting of the eggs, which then develop into cysts in the liver and progress an endless proliferation. Untreated AE has a fatality rate of >90% in humans. Tetraspanins have been identified in Schistosoma and showed potential as the prospective vaccine candidates. In our recent study, we first identified seven tetraspanins in E. multilocularis and evaluated their protective efficacies as vaccines against AE when subcutaneously administered to BALB/c mice. Mucosal immunization of protective proteins is able to induce strong local and systemic immune responses, which might play a crucial role in protecting humans against E. multilocularis infection via the intestine, blood and liver. We focused on Em-TSP3, which achieved significant vaccine efficacy via both s.c. and i.n. routes. The adjuvanticity of nontoxic CpG OND as i.n. vaccine adjuvant was evaluated. The widespread expression of Em-TSP3 in all the developmental stages of E. multilocularis, and the strong local and systemic immune responses evoked by i.n. administration of rEm-TSP3 with CpG OND adjuvant suggest that this study might open the way for developing efficient, nontoxic human mucosal vaccines against AE

Evaluation of Echinococcus multilocularis tetraspanins as vaccine candidates against primary alveolar echinococcosis

Echinococcus multilocularis causes an important zoonotic cestode disease. The metacestocle stage proliferates in the liver of intermediate hosts including human and rodents and forms multiple cysts. Recently, members of a transmembrane protein tetraspanin (TSP) family have been used as vaccines against schistosomosis, or as diagnostic antigens for cysticercosis. In this study, seven tetraspanins of E. multilocularis, designated as TSP1 to TSP7, were evaluated for their protective potential against primary alveolar echinococcosis. The large extracellular loop (LEL) region of these tetraspanins was cloned from a full-length enriched cDNA library of E. multilocularis metacestodes and expressed in Escherichia coli as a fusion protein with thioredoxin. Recombinant TSPs were applied as vaccines against an E. multilocularis primary experimental infection in BALB/c mice. Cyst lesions in the livers of vaccinated and non-vaccinated mice were counted. The cyst lesion reduction rates induced by the seven tetraspanins in vaccinated vis-à-vis non-vaccinated mice were: 87.9%, 65.8%, 85.1%, 66.9%, 73.7%, 72.9% and 37.6%. Vaccination conferred protective rates to mice ranging from 0% (TSP5, 6, 7) to maximally 33% (TSP1, 3). The results indicated that recombinant tetraspanins have varying protective effects against primary alveolar echinococcosis and could be used in vaccine development

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6 × D2)F1 and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host–parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection

Detection of Parasites in the Field: The Ever-Innovating CRISPR/Cas12a

The rapid and accurate identification of parasites is crucial for prompt therapeutic intervention in parasitosis and effective epidemiological surveillance. For accurate and effective clinical diagnosis, it is imperative to develop a nucleic-acid-based diagnostic tool that combines the sensitivity and specificity of nucleic acid amplification tests (NAATs) with the speed, cost-effectiveness, and convenience of isothermal amplification methods. A new nucleic acid detection method, utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, holds promise in point-of-care testing (POCT). CRISPR/Cas12a is presently employed for the detection of Plasmodium falciparum, Toxoplasma gondii, Schistosoma haematobium, and other parasites in blood, urine, or feces. Compared to traditional assays, the CRISPR assay has demonstrated notable advantages, including comparable sensitivity and specificity, simple observation of reaction results, easy and stable transportation conditions, and low equipment dependence. However, a common issue arises as both amplification and cis-cleavage compete in one-pot assays, leading to an extended reaction time. The use of suboptimal crRNA, light-activated crRNA, and spatial separation can potentially weaken or entirely eliminate the competition between amplification and cis-cleavage. This could lead to enhanced sensitivity and reduced reaction times in one-pot assays. Nevertheless, higher costs and complex pre-test genome extraction have hindered the popularization of CRISPR/Cas12a in POCT

Mucosal Adjuvanticity of Fibronectin-Binding Peptide (FBP) Fused with <em>Echinococcus multilocularis</em> Tetraspanin 3: Systemic and Local Antibody Responses

<div><h3>Background</h3><p>Studies have shown that a bacterial fibronectin attachment protein (FAP) is able to stimulate strong systemic and mucosal antibody responses when it is used alone or co-administrated with other antigens (Ags). Thus, it has been suggested to be a promising adjuvant candidate for the development of efficient vaccines. However, the co-administered Ags and FAP were cloned, expressed and purified individually to date. In a recent study, we first evaluated the adjuvanticity of a fibronectin-binding peptide (FBP, 24 amino acids) of <em>Mycobacterium avium</em> FAP fused with <em>Echinococcus multilocularis</em> tetraspanin 3 (Em-TSP3) by detecting systemic and local antibody responses in intranasally (i.n.) immunized BALB/c mice.</p> <h3>Methodology/Principal Findings</h3><p>Em-TSP3 and FBP fragments were linked with a GSGGSG linker and expressed as a single fusion protein (Em-TSP3-FBP) using the pBAD/Thio-TOPO expression vector. BALB/c mice were immunized i.n. with recombinant Em-TSP3-FBP (rEm-TSP3-FBP) and rEm-TSP3+CpG and the systemic and local antibody responses were detected by ELISA. The results showed that both rEm-TSP3-FBP and rEm-TSP3+CpG evoked strong serum IgG (<em>p</em><0.001) and IgG1 responses (<em>p</em><0.001), whereas only the latter induced a high level IgG2α production (<em>p</em><0.001), compared to that of rEm-TSP3 alone without any adjuvant. There were no significant differences in IgG and IgG1 production between the groups. Low level of serum IgA and IgM were detected in both groups. The tendency of Th1 and Th2 cell immune responses were assessed via detecting the IgG1/IgG2α ratio after the second and third immunizations. The results indicated that i.n. immunization with rEm-TSP3-FBP resulted in an increased IgG1/IgG2α ratio (a Th2 tendency), while rEm-TSP3+CpG caused a rapid Th1 response that later shifted to a Th2 response. Immunization with rEm-TSP3-FBP provoked significantly stronger IgA antibody responses in intestine (<em>p</em><0.05), lung (<em>p</em><0.001) and spleen (<em>p</em><0.001) compared to those by rEm-TSP3+CpG. Significantly high level IgA antibodies were detected in nasal cavity (<em>p</em><0.05) and liver (<em>p</em><0.05) samples from both groups when compared to rEm-TSP3 alone without any adjuvant, with no significant difference between them.</p> <h3>Conclusions</h3><p>I.n. administration of rEm-TSP3-FBP can induce strong systemic and mucosal antibody responses in immunized BALB/c mice, suggesting that fusion of Em-TSP3 with FBP is a novel, prospective strategy for developing safe and efficient human mucosal vaccines against alveolar echinococcosis (AE).</p> </div

Solubility and 3-D structure of expressed proteins.

<p>Comparison of hydropathy plots of Em-TSP3 (A) and Em-TSP3-FBP (B). Hydropathy plots of the predicted amino acid sequences were obtained using the Kyte-Doolittle hydropathy plot program (<a href="http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1" target="_blank">http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1</a>). Hydrophobic residues are positive. (C) 3-D structure prediction of Em-TSP3 LEL domain. The amino acid sequence of Em-TSP3 (LEL) was aligned with protein sequences in the structural database using the Phyre (Protein Homology/analogy Recognition Engine) server at Imperial College (<a href="http://www.sbg.bio.ic.ac.uk/phyre/" target="_blank">http://www.sbg.bio.ic.ac.uk/phyre/</a>). The three-dimensional structure was further activated.</p

Cloning and expression of recombinant Em-TSP3-FBP protein.

<p>(A) Synthesis of FBP and Cloning of the LEL and Em-TSP3-FBP. M, marker. Lane 1. Negative control (H₂O). Lane 2. Synthesized FBP (72 bp). Lane 3. PCR product of Em-TSP3 LEL (276 bp). Lane 4. PCR product of Em-TSP3-FBP (366 bp). (B) Expression of recombinant Em-TSP3-FBP (fused with thioredoxin (TRX)). SDS-PAGE gel stained with Coomassie Blue showing purified rEm-TSP3 (Lane 1, 26.2 kDa), Em-TSP3-FBP (Lane 2, 29.3 kDa) and control TRX (Lane 3, 16 kDa). M, the molecular weight marker.</p

List of primers used for fragments amplification.

<p>F = forward;</p><p>R = reverse;</p><p>Tm = melting temperature.</p