Glycolysis- and immune-related novel prognostic biomarkers of Ewing's sarcoma: glucuronic acid epimerase and triosephosphate isomerase 1.

INTRODUCTION: Owing to the poor prognosis of Ewing's sarcoma, reliable prognostic biomarkers are highly warranted for clinical diagnosis of the disease. MATERIALS AND METHODS: A combination of the weighted correlation network analysis and differentially expression analysis was used for initial screening; glycolysis-related genes were extracted and subjected to univariate Cox, LASSO regression, and multivariate Cox analyses to construct prognostic models. The immune cell composition of each sample was analysed using CIBERSORT software. Immunohistochemical analysis was performed for assessing the differential expression of modelled genes in Ewing's sarcoma and paraneoplastic tissues. RESULTS: A logistic regression model constructed for the prognosis of Ewing's sarcoma exhibited that the patient survival rate in the high-risk group is much lower than in the low-risk group. CIBERSORT analysis exhibited a strong correlation of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages (P < 0.05). Immunohistochemical analysis confirmed the study findings. CONCLUSIONS: GLCE and TPI1 can be used as prognostic biomarkers to predict the prognosis of Ewing's sarcoma, and a close association of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages provides a novel approach to the disease immunotherapy.

A Chimera of Th1 Stimulatory Proteins of Leishmania donovani Offers Moderate Immunotherapeutic Efficacy with a Th1-Inclined Immune Response against Visceral Leishmaniasis.

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania (L.) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN-γ and TNF-α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection.

Autoantibodies associated with neuropsychiatric systemic lupus erythematosus: the quest for symptom-specific biomarkers.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs, including the central nervous system. Neuropsychiatric SLE (NPSLE) is a severe and potentially fatal condition. Several factors including autoantibodies have been implicated in the pathogenesis of NPSLE. However, definitive biomarkers of NPSLE are yet to be identified owing to the complexity of this disease. This is a major barrier to accurate and timely diagnosis of NPSLE. Studies have identified several autoantibodies associated with NPSLE;some of these autoantibodies are well investigated and regarded as symptom-specific. In this review, we discuss recent advances in our understanding of the manifestations and pathogenesis of NPSLE. In addition, we describe representative symptom-specific autoantibodies that are considered to be closely associated with the pathogenesis of NPSLE.

Triosephosphate isomerase, carbonic anhydrase, and creatinine kinase-brain isoform are possible antigen targets in patients with achalasia.

BACKGROUND: Idiopathic achalasia is an uncommon esophageal motor disorder. The disease involves interaction between inflammatory and autoimmune responses. However, the antigens related to the disease are still unknown. AIM: To identify the possible antigen targets in muscle biopsies from lower esophageal sphincter (LES) of achalasia patients. METHODS: Esophageal biopsies of patients with type I and type II achalasia and esophagogastric junction outflow obstruction (EGJOO) were analyzed. Lower esophageal sphincter muscle biopsy from a Healthy organ Donor (HD) was included as control for two-dimensional gel electrophoresis. Immunoblotting of muscle from LES lysate with sera of type I, type II achalasia, or type III achalasia, sera of EGJOO and sera of healthy subjects (HS) was performed. The target proteins of the serum were identified by mass spectrometry Matrix-assited laser desorption/ionization time-of-flight (MALDI-TOF). KEY RESULTS: The proteomic map of muscle from LES tissue lysates of type I, and type II achalasia, EGJOO, and HD were analyzed and divided into three important regions. We found a difference in the concentration of certain spots. Further, we observed the serum reactivity of type I achalasia and type II achalasia against 45 and 25 kDa bands of type I achalasia tissue. Serum of type III achalasia and EGJOO mainly recognized 25 kDa band. Bands correspond to triosephosphate isomerase (TPI) (25 kDa), carbonic anhydrase (CA) (25 kDa) and creatinine kinase-brain (CKB) isoform (45 kDa). CONCLUSIONS AND INFERENCES: We identify three antigen targets, TPI, CA, and CKB isoform, which are recognized by sera from patients with achalasia.

Crystal Structure Analysis and Conformational Epitope Mutation of Triosephosphate Isomerase, a Mud Crab Allergen.

The triosephosphate isomerase (TIM), Scy p 8, is a crab allergen and shows cross-reactivity in the shellfish. Here, recombinant Scy p 8 was expressed, and its crystal structure was determined at a resolution of 1.8 Å. The three-dimensional structure of Scy p 8 is primarily composed of a (ß/α)8-barrel motif prototype. Additionally, Scy p 8 showed cross-reactivity with high sequential and secondary structural identity among TIMs from shellfish species. The site-directed mutagenesis of critical amino acids of conformational epitopes was carried out, and the mutants of Trp 168 and Lys 237 to Ala reduced immunoglobulin E (IgE)-binding activity by approximately 30%, compared with wild-type TIM in an inhibition ELISA; however, it still induced basophil activation despite the interpatient variability between patients. These results can help to provide an accurate template for the analysis of the IgE binding and establish meaningful relationships between structure and allergenicity.

Identification of novel antigens for serum IgG diagnosis of human toxoplasmosis.

In the present study, we attempted to identify antigens with high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. We investigated soluble proteins from the tachyzoites of the RH strain of Toxoplasma gondii (T. gondii) and excreted/secreted antigens (ESAs) from the peritoneal protein of T. gondii-infected mice. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis revealed that in both soluble tachyzoite antigens and ESAs, the antigens located between 25 and 35 kDa had high diagnostic sensitivity. Further analysis of antigenic specificity revealed that the antigens located between 25 and 35 kDa were specifically recognized by the sera of toxoplasmosis patients, but other parasitic diseases were not. The protein spots between 25 and 35 kDa were selected after two-dimensional electrophoresis of both soluble tachyzoite antigens and ESAs. GRA2, GRA7, and triosephosphate isomerase (TPI) were successfully characterized from the protein spots using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis. We expressed, purified, and evaluated proteins GRA2, GRA7, and TPI. TPI is a novel antigen with potential for the serological diagnosis of toxoplasmosis, and composite recombinant proteins (TPI, GRA2, and GRA7) have great sera diagnostic value for the detection of the disorder.

Immunotherapeutic potential of Leishmania (Leishmania) donovani Th1 stimulatory proteins against experimental visceral leishmaniasis.

An effective therapeutic vaccination strategy is required for controlling visceral leishmaniasis (VL), a fatal systemic disease, through boosting the immunosuppressed state in Leishmania-infected individuals, as the majority of them living in the endemic regions exhibit either subclinical or asymptomatic infection which further often develops into a full-blown disease. Previously in our laboratory, several Th1 stimulatory recombinant proteins were successfully cloned, purified and assessed for their prophylactic efficacy against Leishmania challenge. Due to their immunostimulatory property, these proteins are needed to be evaluated for their immunotherapeutic potential in Leishmania-infected hamsters. Four proteins namely, aldolase, enolase, p45 and triose phosphate isomerase were taken up to immunize animals at different doses (50, 25 and 12.5 µg/animal). Immunization with lower doses of aldolase and enolase, i.e., 25 and 12.5 µg showed a significant decline (∼60%) in parasitic load along with an enhanced cellular immune response. These findings indicate that vaccination with above -stated Th1 stimulatory proteins is an effective immunotherapeutic approach against experimental VL. However, their efficacies may further be improved in combination with known therapeutic regimens or immunomodulators.

Association of anti-triosephosphate isomerase antibodies with aseptic meningitis in patients with neuropsychiatric systemic lupus erythematosus.

Autoantibodies to triosephosphate isomerase (TPI), which is an important glycolytic enzyme in red blood cells and neuronal cells, have been reported to be associated with neuropsychiatric systemic lupus erythematosus (NPSLE) pathogenesis. However, the clinical features regarding anti-TPI antibody (anti-TPI)-positive NPSLE are not yet known. The aim of this study is to investigate the clinical features of anti-TPI-positive NPSLE patients using anti-TPI index values determined by enzyme-linked immunosorbent assay (ELISA). Thirty-one NPSLE patients treated in our department were included in this study. Serum samples were collected, and serum anti-TPI titers were measured by ELISA. The anti-TPI index values were defined as follows: (OD405 of samples - OD405 of negative control)/(OD405 of positive control - OD405 of negative control) × 100. Anti-TPI index values greater than 2 standard deviations above the mean of healthy controls were regarded as positive. The clinical features of anti-TPI-positive and anti-TPI-negative NPSLE were compared. Ten of the 31 NPSLE patients had anti-TPI positivity (32.3%). The clinical features of anti-TPI-positive NPSLE were comparable with those of anti-TPI-negative NPSLE, except for a higher frequency of aseptic meningitis (p = 0.027) and a lower frequency of acute confusional state (P = 0.026). Laboratory data in patients with anti-TPI-positive NPSLE showed significantly higher serum IgG levels. Furthermore, anti-TPI index values positively correlated with serum IgG levels. Our study indicates that serum anti-TPI increases in the presence of elevated IgG levels and can be associated with the pathogenesis of aseptic meningitis in NPSLE.

Evaluation of protective efficacy induced by different heterologous prime-boost strategies encoding triosephosphate isomerase against Schistosoma japonicum in mice.

BACKGROUND: In China, schistosomiasis japonica is a predominant zoonotic disease, and animal reservoir hosts in the environment largely sustain infections. The development of transmission-blocking veterinary vaccines is urgently needed for the prevention and efficient control of schistosomiasis. Heterologous prime-boost strategy is more effective than traditional vaccination and homologous prime-boost strategies against multiple pathogens infection. In the present study, to further improve protective efficacy, we immunized mice with three types of heterologous prime-boost combinations based on our previously constructed vaccines that encode triosphate isomerase of Schistosoma japonicum, tested the specific immune responses, and evaluated the protective efficacy through challenge infection in mice. METHODS: DNA vaccine (pcDNA3.1-SjTPI.opt), adenoviral vectored vaccine (rAdV-SjTPI.opt), and recombinant protein vaccine (rSjTPI) were prepared and three types of heterologous prime-boost combinations, including DNA i.m. priming-rAdV i.m. boosting, rAdV i.m. priming-rAdV s.c. boosting, and rAdV i.m. priming-rSjTPI boosting strategies, were carried out. The specific immune responses and protective efficacies were evaluated in BALB/c mice RESULTS: Results show that different immune profiles and various levels of protective efficacy were elicited by using different heterologous prime-boost combinations. A synergistic effect was observed using the DNA i.m. priming-rAdV i.m. boosting strategy; however, its protective efficacy was similar to that of rAdV i.m. immunization. Conversely, an antagonistic effect was generated by using the rAd i.m. priming-s.c. boosting strategy. However, the strategy, with rAdV i.m. priming- rSjTPI s.c. boosting, generated the most optimal protective efficacy and worm or egg reduction rate reaching up to 70% in a mouse model. CONCLUSIONS: A suitable immunization strategy, rAdV i.m. priming-rSjTPI boosting strategy, was developed, which elicits a high level of protective efficacy against Schistosoma japonicum infection in mice.

Identification of triosephosphate isomerase as a novel allergen in Octopus fangsiao.

Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A71G74S69D75T73F72V67) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis.

Triosephosphate Isomerase and Filamin C Share Common Epitopes as Novel Allergens of Procambarus clarkii.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

[Preliminary research on prokaryotic expression and immune protection of triosephosphate isomerase of Toxoplasma gondii].

OBJECTIVE: To study the prokaryotic expression and immune protection of triosephosphate isomerase (TPI) of Toxoplasma gondii in mice. METHODS: Total RNA was extracted from toxoplasma tachyzoites, and TPI fragment was amplified by PCR and cloned into the prokaryotic expression vector pET-28a (+). The target protein was induced with IPTG and purified by Ni-NTA affinity chromatography. The mice were immunized 4 times by emulsified TPI with adjuvant, and the last time was the strengthen immunization. At the same time, an adjuvant group and a normal group were set as controls. The blood samples were got from the tail vein of the mice, and the serum antibody titres were detected. All the mice were challenged with 400 toxoplasma tachyzoites to observe the survival time. RESULTS: The TPI gene was amplified from T. gondii cDNA by PCR. The recombinant vector TPI/pET-28a (+) was usefully constructed, and the TPI protein was expressed and purified. The serum antibody titre could be more than 100 thousand. After infected with toxoplasma tachyzoites, the survival time of the mice in the experimental group was longer than that of the mice in the control groups. CONCLUSIONS: The TPI protein of T. gondii could trigger the immunoprotection against T. gondii challenge in the mice.

In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

SjTat-TPI facilitates adaptive T-cell responses and reduces hepatic pathology during Schistosoma japonicum infection in BALB/c mice.

BACKGROUND: Schistosomiasis is a kind of parasitic zoonoses which causes serious damage to public health and social development. China is one of the countries most affected by Schistosoma japonicum and an effective vaccine is still needed. In this study, we adopted Tat-mediated protein transduction technology to investigate the impact of different antigen presented approaches on host's immune response and the potential protection against Schistosoma japonicum infection. RESULTS: We successfully constructed the recombinant S. japonicum triosephosphate isomerase, Tat-TPI, as a vaccine candidate. Whether injected with Tat-TPI in foot pad or vaccinated with Tat-TPI in the back subcutaneously for three times, the draining popliteal lymph nodes and spleen both developed a stronger CD8(+)T response (Tc1) in mice. Not only that, but it also helped CD4(+)T cells to produce more IFN-γ than TPI immunisation. In addition, it could boost IgG production, especially IgG1 subclass. Most importantly, Tat-TPI immunisation led to the significant smaller area of a single egg granuloma in the livers as compared with TPI-vaccinated or control groups. However, the anti-infection efficiency induced by Tat-TPI was still restricted. CONCLUSION: This study indicated that immunisation with Tat-fused TPI could contribute to enhance CD4(+)T-cell response and decrease hepatic egg granulomatous area after S. japonicum infection though it did not achieve our expected protection against Schistosoma japonicum infection. The optimal vaccine strategy warrants further research.

Enhancement of protective efficacy through adenoviral vectored vaccine priming and protein boosting strategy encoding triosephosphate isomerase (SjTPI) against Schistosoma japonicum in mice.

BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease; developing transmission blocking veterinary vaccines are urgently needed for the prevention and control of schistosomiasis in China. Heterologous prime-boost strategy, a novel vaccination approach, is more effective in enhancing vaccine efficacy against multiple pathogens. In the present study, we established a novel heterologous prime-boost vaccination strategy, the rAdV-SjTPI.opt intramuscular priming and rSjTPI subcutaneous boosting strategy, and evaluated its protective efficacy against Schistosoma japonicum in mice. METHODOLOGY/PRINCIPAL FINDINGS: Adenoviral vectored vaccine (rAdV-SjTPI.opt) and recombinant protein vaccine (rSjTPI) were prepared and used in different combinations as vaccines in a mouse model. The specific immune responses and protective efficacies were evaluated. Furthermore, the longevity of protective efficacy was also determined. Results showed that the rAdV-SjTPI.opt priming-rSjTPI boosting strategy elicited higher levels of specific IgG responses and broad-spectrum specific cellular immune responses. The protective efficacy could reach up to nearly 70% and 50% of protection could be observed at 10 weeks after the last immunization in mice. CONCLUSIONS/SIGNIFICANCE: The rAdV-SjTPI.opt intramuscular priming-rSjTPI subcutaneous boosting vaccination strategy is a novel, highly efficient, and stable approach to developing vaccines against Schistosoma japonicum infections in China.

Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity.

Previous studies demonstrated that antibodies against triosephosphate isomerase of Taenia solium (TTPI) can alter its enzymatic catalysis. In the present study, we used antibodies produced against the NH2-terminal region of TTPI (1/3NH2TTPI) and the phage display technology to find target regions to inhibit TTPI activity. As a first step, we obtained polyclonal antibodies against non-conserved regions from the 1/3NH2TTPI, which had an inhibitory effect of about 74 % on catalytic activity. Afterward, they were used to screen a library of phage-displayed dodecapeptides; as a result, 41 phage mimotope clones were isolated and grouped according to their amino acid sequence, finding the consensus A1 (VPTXPI), A2 (VPTXXI), B (LTPGQ), and D (DPLPR). Antibodies against selected phage mimotope clones were obtained by rabbit's immunization; these ones clearly recognized TTPI by both Western blot and ELISA. However, only the mimotope PDTS16 (DSVTPTSVMAVA) clone, which belongs to the VPTXXI consensus, raised antibodies capable of inhibiting the TTPI catalytic activity in 45 %. Anti-PDTS16 antibodies were confronted to several synthetic peptides that encompass the 1/3NH2TTPI, and they only recognized three, which share the motif FDTLQK belonging to the helix-α1 in TTPI. This suggests that this motif is the main part of the epitope recognized by anti-PDTS16 antibodies and revealed its importance for TTPI catalysis.

Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody in vaccinated animals. These observations indicated that vaccine(s) based on combination of HSP70 with Th1-stimulatory protein(s) may be a viable proposition against intracellular pathogens.

Construction and evaluation of replication-defective recombinant optimized triosephosphate isomerase adenoviral vaccination in Schistosoma japonicum challenged mice.

Schistosomiasis is an endemic, zoonotic parasitic disease that remains a public health concern in China. Development of transmission blocking veterinary vaccines against Schistosoma japonicum infection is urgently needed. Replication-defective adenoviral vector is an efficient vaccine delivery system that has been widely used. Its use is associated with high levels of gene insertion and expression. It is easy to construct and prepare, and is safe. It is not known whether this delivery system can improve the protective effect of schistosome vaccination. Triosephosphate isomerase from S. japonicum (SjTPI) is a promising vaccine candidate. Thus far it has induced only partial protection in animal models and needs to be further enhanced to be effective. We constructed a replication-defective adenoviral vector-based vaccine with optimized SjTPI (rAdV-SjTPI.opt). The specific immune responses and protective efficiency in mice were evaluated. Results showed that intramuscular rAdV-SjTPI.opt induced Th1 biased immune responses in the host, while subcutaneous rAdV-SjTPI.opt induced Th2 predominant immune responses. Oral rAdV-SjTPI.opt induced low levels of immune responses and no significant protection. Intramuscular rAdV-SjTPI.opt provided a consistent and repeatable higher protective effect in mice (more than 50%). These findings may be due to the associated higher levels of specific Th1, antibody responses and partially lower level of IL-17A. This report provides a foundation for developing transmission-blocking veterinary vaccines in larger animals.

Triose phosphate isomerase from the blood fluke Schistosoma mansoni: biochemical characterisation of a potential drug and vaccine target.

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0 °C). The steady-state kinetic parameters are high (Km for dihydroxyacetone phosphate is 0.51 mM; Km for glyceraldehyde 3-phosphate is 1.1 mM; kcat for dihydroxyacetone phosphate is 7800 s(-1) and kcat for glyceraldehyde 3-phosphate is 6.9s(-1)).

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.