Worm Reduction Research Articles

Acute Toxicity and IN VIVO Antischistosomal Effects of Entada africana Guill & Perott (Fabaceae) Methanol Stem Bark Extract in Albino Mice

The study was aimed at determining the acute toxicity and schistosomicidal effects of methanol extract (ME) of Entada africana (E. africana), in albino mice. Fresh stem bark of E. africana was shade-dried and extracted by Soxhlet extraction. Acute toxicity was carried out on 6 groups of mice (n=3) of 6-week-old, sizes 27-30g; to determine the therapeutic index, piloerection signs and possible mortality (LD50) in 24h exposure period. Cercaria from B. globosus snails were shed under a 100W bulb for 2 hours. In vivo antischistosomal assays was by used of 6-weeks old (27-30g) mice. Set I (n=10) involved investigation on schistosomules infection in the mice, 3-7 days post infection (PI). Set II (n=10) was infected and treated with E. africana ME extract, at concentrations 10-100mg/l, 35th day, PI for 5 consecutive days. Set III (n=10), were infected and treated with praziquantel– PZQ (positive control). While set IV (n=10) were infected and untreated (negative control). All were sacrificed, 75th day of PI. Data were analyzed by GraphPad® prism 8.4, version 2020. Findings revealed that E. africana ME did not generate any harmful or clinical effect within 24h, post administration; and there were no obvious reactions such as paw licking, stooling, piloerection or immediate death, except at phase II, 5000mg/kg bwt, (1⁄3) being the highest dosage. Worm burden decreased with increase in concentrations, especially at 70-100mg/l. Lower concentrations (10mg/l), had high worm population (76.00±21.28). Infected and untreated group had highest worm population (189.67±32.52). E. africana ME stem bark extract, showed antischistosomal effect and worm reduction in a dose- dependent pattern.

In vivo anti-schistosomal activity of the methanol extracts from Searsia longipes and Lannea schimperi

Schistosomiasis is a disease caused by the flat worms under the genus Schistosoma. The disease is prominent in tropical and sub tropical countries and it is manifested in two forms; the acute and the chronic form. Treatment and control of the schistosomiasis is constrained with various factors including immerging worm resistance and selective efficacy of the current recommended drug of choice. This therefore calls for the search of new approaches to offset the problems. The aim of this study was to investigate the efficacy of the methanolic extract from Searsia longipes and Lannea schimperi against Schistosoma mansoni by using animal model. Swiss albino mice were used for the efficacy testing, whereby, for each extract, 5 groups of mice were used, and each contained 5 mice. Three different doses were administered to three groups, whereas the remaining two groups were used as negative and positive control arms. Efficacies were assessed based on the reduction of the worm eggs in the faeces and organs, body weight gain, loss of liver weight, as well as reduction of worm burden. Both extracts demonstrated significant egg and worm reduction, which was directly proportional to the dose increment. At the highest dose used, Searsia longipes expressed the higher percentage egg reduction (73.33 %), whereas Lannea schimperi demonstrated the higher worm burden reduction (68.53 %). The present study provides strong evidence of the anti-schistosomal activity of the extracts from both S. longipes and L. schimperi. These findings are a significant step forward, suggesting that these plants could be a promising alternative medicine for the treatment of schistosomiasis. However, further investigations are warranted to isolate the compound responsible for this activity and to assess the sub-acute toxicity of the plant extracts. This next phase of research is crucial for advancing our understanding and potential use of these plant extracts.

Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

Impact of atorvastatin and mesenchymal stem cells combined with ivermectin on murine trichinellosis

Trichinellosis is one of the global food-borne parasitic diseases that can cause severe tissue damage. The traditionally used drugs for the treatment of trichinellosis have limited efficacy against the encysted larvae in the muscular phase of the disease. Therefore, this study aimed to evaluate the role of atorvastatin and mesenchymal stem cells combined with ivermectin against different phases of Trichinella in experimentally infected mice. A total of 120 male Swiss albino mice were divided into two major groups (n = 60 of each), intestinal and muscular phases. Then, each group was subdivided into 10 subgroups (n = 6); non-infected control, infected non-treated control, infected ivermectin treated, infected atorvastatin treated, infected mesenchymal stem cells treated, infected combined ivermectin and atorvastatin treated, infected combined mesenchymal stem cells and ivermectin treated, infected combined mesenchymal stem cells and atorvastatin treated, infected combined mesenchymal stem cells and a full dose of (ivermectin and atorvastatin) treated, and infected combined mesenchymal stem cells and half dose of (ivermectin and atorvastatin) treated. Mice were sacrificed at days 5 and 35 post-infection for the intestinal and muscular phases, respectively. The assessment was performed through many parameters, including counting the adult intestinal worms and muscular encysted larvae, besides histopathological examination of the underlying tissues. Moreover, a biochemical assay for the inflammatory and oxidative stress marker levels was conducted. In addition, levels of immunohistochemical CD31 and VEGF gene expression as markers of angiogenesis during the muscular phase were investigated. The combined mesenchymal stem cells and atorvastatin added to ivermectin showed the highest significant reduction in adult worms and encysted larvae counts, the most noticeable improvement of the histopathological changes, the most potent anti-inflammatory (lowest level of IL-17) and anti-angiogenic (lowest expression of CD31 and VEGF) activities, and also revealed the highly effective one to relieve the oxidative stress (lowest level of SOD, GSH, and lipid peroxidase enzymes). These observed outcomes indicate that adding mesenchymal stem cells and atorvastatin to ivermectin synergistically potentiates its therapeutic efficacy and provides a promising candidate against trichinellosis.

Protective efficacy of six recombinant proteins as vaccine candidates against Echinococcus granulosus in dogs.

Cystic echinococcosis (CE) is caused by the infection of Echinococcus granulosus sensu lato (E. granulosus s.l.), one of the most harmful zoonotic helminths worldwide. Infected dogs are the major source of CE transmission. While praziquantel-based deworming is a main measure employed to control dog infections, its efficacy is at times compromised by the persistent high rate of dog re-infection and the copious discharge of E. granulosus eggs into the environment. Therefore, the dog vaccine is a welcome development, as it offers a substantial reduction in the biomass of E. granulosus. This study aimed to use previous insights into E. granulosus functional genes to further assess the protective efficacy of six recombinant proteins in dogs using a two-time injection vaccination strategy. We expressed and combined recombinant E. granulosus triosephosphate isomerase (rEgTIM) with annexin B3 (rEgANXB3), adenylate kinase 1 (rEgADK1) with Echinococcus protoscolex calcium binding protein 1 (rEgEPC1), and fatty acid-binding protein (rEgFABP) with paramyosin (rEgA31). Beagle dogs received two subcutaneous vaccinations mixed with Quil-A adjuvant, and subsequently orally challenged with protoscoleces two weeks after booster vaccination. All dogs were sacrificed for counting and measuring E. granulosus tapeworms at 28 days post-infection, and the level of serum IgG was detected by ELISA. Dogs vaccinated with rEgTIM&rEgANXB3, rEgADK1&rEgEPC1, and rEgFABP-EgA31 protein groups exhibited significant protectiveness, with a worm reduction rate of 71%, 57%, and 67%, respectively, compared to the control group (P < 0.05). Additionally, the vaccinated groups exhibited an inhibition of worm growth, as evidenced by a reduction in body length and width (P < 0.05). Furthermore, the level of IgG in the vaccinated dogs was significantly higher than that of the control dogs (P < 0.05). These verified candidates may be promising vaccines for the prevention of E. granulosus infection in dogs following two injections. The rEgTIM&rEgANXB3 co-administrated vaccine underscored the potential for the highest protective efficacy and superior protection stability for controlling E. granulosus infections in dogs.

Evaluation of the immunoprotective effects of eight recombinant proteins from Baylisascaris schroederi in mice model

BackgroundBaylisascaris schroederi is the most common and harmful intestinal parasitic nematode of giant pandas, causing ascariasis. Although drug deworming is the main measure to control ascariasis in captive giant pandas, prolonged and repeated use of deworming drugs might induce resistance in nematodes and drug residues in giant pandas. Therefore, developing a safe and effective vaccine might provide a novel strategy to prevent ascariasis in captive giant pandas.MethodsFour highly expressed secretome genes encoding excretory and secretory proteins of B. schroederi, including transthyretin-like protein 46 (BsTLP), uncharacterized protein (BsUP), hypothetical protein 1 (BsHP1), and hypothetical protein 2 (BsHP2) and four functional genes [(encoding Galectin (BsGAL), glutathione S-transferase (BsGST), fatty acid-binding protein (BsFABP), and thioredoxin peroxidase (BsTPX)] were identified based on genome and transcriptome databases of B. schroederi and used to construct recombinant proteins via prokaryotic expression. Kunming mice were vaccinated subcutaneously twice with the recombinant proteins (50 μg/mouse) mixed with Quil A adjuvant with a 2-week interval and then orally challenged with 3000 infective eggs. The immunoprotective effects of the eight recombinant proteins on mice were assessed comprehensively using surface lesion histology scores of the mouse liver and lung, larval worm reduction, serum antibody levels (IgG, IgE, IgA, IgG1, and IgG2a), and cytokine production [interferon gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-5, and IL-10].ResultsMice vaccinated with recombinant (r)BsUP (76.5%), rBsGAL (74.7%), and rBsHP2 (71.5%) showed a significant (P < 0.001) reduction in the larval worm rate compared with that in the adjuvant control. Besides, the surface lesions in the liver and lung of the vaccinated mice were alleviated. Serum levels of total IgG, IgE, IgA, IgG1, IgG2a, and cytokines, including IL-10, IL-5, and IFN-γ, were significantly higher (P < 0.001) than those in the control group.ConclusionsThe results showed that candidate three vaccines (rBsUP, rBsGAL, and rBsHP2) could provide effective protection against egg infection in mice associated with a mixed Th1/2-type immune response.Graphical

Small change, big difference: A promising praziquantel derivative designated P96 with broad-spectrum antischistosomal activity for chemotherapy of schistosomiasis japonica.

Praziquantel (PZQ) has been the first line antischistosomal drug for all species of Schistosoma, and the only available drug for schistosomiasis japonica, without any alternative drugs since the 1980s. However, PZQ cannot prevent reinfection, and cannot cure schistosomiasis thoroughly because of its poor activity against juvenile schistosomes. In addition, reliance on a single drug is extremely dangerous, the development and spread of resistance to PZQ is becoming a great concern. Therefore, development of novel drug candidates for treatment and control of schistosomiasis is urgently needed. One of the PZQ derivative christened P96 with the substitution of cyclohexyl by cyclopentyl was synthesized by School of Pharmaceutical Sciences of Shandong University. We investigated the in vitro and in vivo activities of P96 against different developmental stages of S. japonicum. Parasitological studies and scanning electron microscopy were used to study the primary action characteristics of P96 in vitro. Both mouse and rabbit models were employed to evaluate schistosomicidal efficacy of P96 in vivo. Besides calculation of worm reduction rate and egg reduction rate, quantitative real-time PCR was used to evaluate the in vivo antischistosomal activity of P96 at molecular level. In vitro, after 24h exposure, P96 demonstrated the highest activities against both juvenile and adult worm of S. japonicum in comparison to PZQ. The antischistosomal efficacy was concentration-dependent, with P96 at 50μM demonstrating the most evident schistosomicidal effect. Scanning electron microscopy demonstrated that P96 caused more severe damages to schistosomula and adult worm tegument compared to PZQ. In vivo, our results showed that P96 was effective against S. japonicum at all developmental stages. Notably, its efficacy against young stage worms was significantly improved compared to PZQ. Moreover, P96 retained the high activity comparable to PZQ against the adult worm of S. japonicum. P96 is a promising drug candidate for chemotherapy of schistosomiasis japonica, which has broad spectrum of action against various developmental stage, potentially addressing the deficiency of PZQ. It might be promoted as a drug candidate for use either alone or in combination with PZQ for the treatment of schistosomiasis.

Optimising mopane worm (Gonimbrasia belina) processing for improved nutritional and microbial quality.

Mopane worms (Gonimbrasia belina) is an important source of food and contribute to the nutrition of people who consume them. However, the traditional processing methods may have detrimental effects on the nutritional value and should also guarantee microbial quality. In this study, the nutritional composition and microbial quality of mopane worms processed under different boiling time (0-20 min) and drying temperature (40-60 °C) conditions were investigated and optimised using response surface methodology. An increase in the boiling time at the lowest drying temperature resulted in an increase in protein content and reduction in coliform counts. The optimum conditions of boiling for 20 min and drying at 40 °C resulted in mopane worms with a protein content of 49.4% DW and coliform counts <1.5 log cfu/g. In addition, high concentrations of crude fibre (13.6% DW) and fat (20.2% DW), as well as Fe (19.0 mg/100 g) and Zn (17.9 mg/100 g) were also recorded. A decrease in the total bacterial count, Escherichia coli and yeasts and moulds at the boiling time ≥20 min irrespective of the drying temperature suggested that exposure to heat reduced the microbial growth and contamination. Reduction of the mopane worms' moisture content (<7%) due to drying further slowed down the rate of microbial growth. The optimal processing conditions (boiling for 20 min and drying at 40 °C) are recommended for pretreatment of mopane worms prior to further processing into various products.

Praziquantel promotes protection against Schistosoma japonicum infection in mice

Praziquantel (PZQ) is the first line drug for the treatment of schistosomiasis. Several studies have confirmed that PZQ regulates host immunity, and we have recently found that pretreatment with PZQ enhances resistance against Schistosoma japonicum infection in buffaloes. We speculate that PZQ induces physiological changes in mice that prevent S. japonicum infection. To test this hypothesis and provide a practical measure to prevent S. japonicum infection, we determined the effective dose (the minimum dose), protection period and onset time of protection by comparing the worm burden, female worm burden and egg burden in PZQ-pretreated mice and blank control mice. Morphological differences between parasites were observed by measuring the total worm length, oral sucker, ventral sucker and ovary. The levels of cytokines, nitrogen monoxide (NO), 5-hydroxytryptamine (5-HT) and specific antibodies were measured using kits or soluble worm antigens. Hematological indicators on day 0 were analyzed in mice that received PZQ on days -15, -18, -19, -20, -21 and -22. The PZQ concentrations in plasma and blood cells were monitored using high performance liquid chromatography (HPLC). The effective dose was found to be two oral administrations (interval of 24 h) at 300 mg/kg body weight (BW) or one injection at 200 mg/kg BW, and the protection period of PZQ injection was 18 days. The optimal preventive effect was observed at two days post-administration, with a >92% worm reduction rate and significant worm reduction until 21 days after administration. Adult worms from PZQ-pretreated mice were runtish showing a shorter length, smaller organs and fewer eggs in the uteri of females. Detection of cytokines, NO, 5-HT and hematological indicators showed that PZQ induced immune-physiological changes, including higher levels of NO, IFN-γ and IL-2, and a lower level of TGF-β. No significant difference in the anti-S. japonicum specific antibody levels was observed. The PZQ concentrations in plasma and blood cells 8 and 15 days post-administration were lower than the detection limit. Our results confirmed that pretreatment with PZQ promotes the protection of mice against S. japonicum infection within 18 days. Although we observed some immune-physiological changes in the PZQ-pretreated mice, the exact mechanisms involved in the preventive effect require further study.

Oral vaccination of mice with attenuated Salmonella encoding Trichinella spiralis calreticulin and serine protease 1.1 confers protective immunity in BALB/c mice.

Trichinella spiralis is a foodborne parasitic nematode which is a serious risk to meat safety. Development of anti-Trichinella vaccine is needed to control Trichinella infection in food animals. In this study, two novel T. spiralis genes (calreticulin and serine protease 1.1) in combination were used to construct oral DNA vaccines, and their induced protective immunity was evaluated in a murine model. TsCRT+TsSP1.1, TsCRT and TsSP1.1 DNA were transformed into attenuated Salmonella typhimurium ΔcyaSL1344. Oral vaccination of mice with TsCRT+TsSP1.1, TsCRT and TsSP1.1 DNA vaccines elicited a gut local mucosal sIgA response and systemic Th1/Th2 mixed response. Oral vaccination with TsCRT+TsSP1.1 induced obviously higher level of serum specific antibodies, mucosal sIgA and cellular immune response than either of single TsCRT or TsSP1.1 DNA vaccination. Oral vaccination of mice with TsCRT+TsSP1.1 exhibited a 53.4% reduction of enteral adult worms and a 46.05% reduction of muscle larvae, conferred a higher immune protection than either of individual TsCRT (44.28 and 42.46%) or TsSP1.1 DNA vaccine (35.43 and 29.29%) alone. Oral vaccination with TsCRT+TsSP1.1, TsCRT and TsSP1.1 also obviously ameliorated inflammation of intestinal mucosa and skeletal muscles of vaccinated mice after challenge. TsCRT and TsSP1.1 might be regarded the novel potential targets for anti-Trichinella vaccines. Attenuated Salmonella-delivered DNA vaccine provided a prospective approach to control T. spiralis infection in food animals.

Extracellular vesicles from Trichinella spiralis: Proteomic analysis and protective immunity.

Trichinella spiralis (T. spiralis) derived extracellular vesicles (EVs) have been proposed to play a key role in regulating the host immune responses. In this study, we provided the first investigation of EVs proteomics released by T. spiralis muscle larvae (ML). T. spiralis ML EVs (Ts-ML-EVs) were successfully isolated and characterized by transmission electron microscopy (TEM) and western blotting. Using liquid chromatograph mass spectrometer (LC-MS/MS) analysis, we identified 753 proteins in the Ts-ML-EVs proteome and annotated by gene ontology (GO). These proteins were enriched in different categories by GO, kyoto encyclopedia of genes and genomes (KEGG) and domain analysis. GO enrichment analysis indicated association of protein deglutathionylation, lysosomal lumen and serine-type endopeptidase inhibitor activity with proteins which may be helpful during parasite-host interaction. Moreover, KEGG enrichment analysis revealed involvement of Ts-ML-EVs proteins in other glycan degradation, complement and coagulation cascades, proteasome and various metabolism pathways. In addition, BALB/c mice were immunized by subcutaneous injection of purified Ts-ML-EVs. Ts-ML-EVs group demonstrated a 23.4% reduction in adult worms and a 43.7% reduction in ML after parasite challenge. Cellular and humoral immune responses induced by Ts-ML-EVs were detected, including the levels of specific antibodies (IgG, IgM, IgE, IgG1 and IgG2a) as well as cytokines (IL-12, IFN-γ, IL-4 and IL-10) in serum. The results showed that Ts-ML-EVs could induce a Th1/Th2 mixed immune response with Th2 predominant. This study revealed a potential role of Ts-ML-EVs in T. spiralis biology, particularly in the interaction with host. This work provided a critical step to against T. spiralis infection based on Ts-ML-EVs.

Application of Urine and Copro Antigen Assays after Primary Infection and Drug Treatment in an Experimental Opisthorchiasis Animal Model.

Infection by Opisthorchis viverrini causes significant health problems, including cholangiocarcinoma (CCA); thus control and elimination of this trematode is an important strategy for the reduction of CCA. Currently, urine and copro antigen detection is more sensitive than parasitological examination of the feces for the diagnosis of opisthorchiasis. Given limitations in human studies, we used an animal model to quantify the parasite antigen profiles in urine and feces in O. viverrine-infected hamsters, and postdrug treatment. The positive detections of O. viverrini antigen began from week 1 in urine and week 2 in feces after infection until week 28 of the study. The recoveries of O. viverrini worms were detected starting from week 1 and eggs of O. viverrini were detected in feces from week 3 after infection and remained detectable throughout the study period. There was a significant positive correlation of urine and copro antigen levels with the number of fecal egg counts (P < 0.01) and worm recovery (P < 0.01). In the drug-treatment experiment, treatment of infected hamsters with praziquantel significantly reduced worm burden, fecal egg output, and antigen in urine and feces compared with the untreated controls (P < 0.001). At 4 weeks posttreatment, the egg and worm reduction rates were 100% and 95.5%, respectively. The positive antigen detections in urine and feces corresponded with partial worm clearance from praziquantel treatment. This study demonstrated a direct link of urine and copro antigen tests with worms infecting the liver thereby reaffirming the reliability of urine and copro antigen assay in opisthorchiasis diagnosis.

The Potential Role of MicroRNA-124-3p in Growth, Development, and Reproduction of Schistosoma japonicum.

The microRNA‐124‐3p plays an important role in regulating development and neurogenesis. Previous microRNA sequencing analyses of Schistosoma japonicum revealed sja-miR-124-3p differential expression patterns in schistosomes from different hosts and at different developmental stages. This study explores the regulatory role of sja-miR-124-3p in S. japonicum development and reproduction. Quantitative reverse-transcription PCR (qRT-PCR) showed that the expression level of sja-miR-124-3p in S. japonicum from resistant hosts, such as Microtus fortis, and unsuitable hosts, such as rats and water buffalo, was significantly higher than that in mice and yellow cattle at the same developmental stage. Overexpressing sja-miR-124-3p in infected mice led to a hepatic egg reduction rate of 36.97%, smaller egg granulomas in the livers, increased liver weight, subsided hepatocyte necrosis, and diminished inflammatory cell infiltration. The width of female worms increased but decreased in males. The vitelline cells were irregular, swollen, or fused. The teguments and ventral sucker of males and females were swollen and broken, but the morphological changes were particularly notable in males. qRT-PCR and dual-luciferase reporter assay system were used to confirm the in-silico-predicted target genes, S. japonicum DEAD-box ATP-dependent RNA helicase 1 (sjDDX1) and DNA polymerase II subunit 2 (sjPOLE2). Our results showed that RNA interference (RNAi)-mediated sjDDX1 silencing in mice provided a 24.55% worm reduction rate and an 18.36% egg reduction rate, but the difference was not significant (p > 0.05). Thus, our findings suggest that sja-miR-124-3p has an important role in growth, development, and reproduction in S. japonicum. All these results will greatly contribute toward providing important clues for searching vaccine candidates and new drug targets against schistosomiasis.

Development of a peptide vaccine against hookworm infection: Immunogenicity, efficacy, and immune correlates of protection

Approximately 400 million individuals are infected with hookworms globally. Protective vaccines are needed to prevent reinfections, which often occur after drug treatment in endemic areas. Ideal vaccines are highly efficacious and well tolerated, and do not present risks to patient safety. Peptide vaccines can generate specific, highly protective responses because they focus on minimal antigenic target(s) with a specific immunoprotective mechanism. Necator americanus aspartyl protease 1 (Na-APR-1) is one of the most promising hookworm vaccine antigens. The neutralizing epitope p3 (TSLIAGPKAQVEAIQKYIGAEL), together with universal the TH epitope P25 (KLIPNASLIENCTKAEL), has been used previously to produce peptide vaccines and was found to protect BALB/c mice against rodent hookworm infections, resulting in worm burden reductions of up to 98%. However, because of extensive digestion in the gastrointestinal tract, large oral vaccination doses were necessary to achieve this level of efficacy. We sought to overcome the limitations of oral vaccine delivery and to investigate protective efficacy and immune correlates of protection. Herein, we examined 5 different peptide vaccines following intraperitoneal injection, to compare their efficacy with that of the clinical protein antigen APR-1. BALB/c mice were immunized with p3-P25-based antigen that was adjuvanted with (1) lipid core peptide, (2) polymethyl methacrylate, (3) linear polyleucine, and (4) branched polyleucine (BL10), or with (5) CpG/aluminum hydroxide adjuvant (alum)-adjuvanted control and protein-based (6) CpG/alum-adjuvanted Na-APR-1. The mice sera, saliva, and feces were sampled for immune response evaluation. The immunized mice were further challenged via hookworm larvae infection, and protection was evaluated by conducting intestinal hookworm counts. BL10 and lipid core peptide generated the highest serum anti-Na-APR-1 IgG and fecal anti-APR-1 IgG titers, but only BL10 generated significant fecal anti-Na-APR-1 IgA titers. Upon challenge, immunization with CpG/alum-adjuvanted p3-P25, BL10, and lipid core peptide provided the highest worm burden reductions of 75%, 77%, and 59%, respectively, whereas the group immunized with Na-APR-1 had only modest worm reduction of 26%. The relationships between serum anti-Na-APR-1 IgG, fecal anti-Na-APR-1 IgA and IgG, and worm burden reduction were established with R2 values greater than or equal to 0.9, and the crucial role of both anti-Na-APR-1 IgG and IgA responses was identified. We demonstrated for the first time that p3-based vaccine candidates are safer and can deliver higher protection against hookworm infection compared with the clinical vaccine candidate, Na-APR-1.

Mucosal Vaccination With Recombinant Tm-WAP49 Protein Induces Protective Humoral and Cellular Immunity Against Experimental Trichuriasis in AKR Mice.

Trichuriasis is one of the most common neglected tropical diseases of the world’s poorest people. A recombinant vaccine composed of Tm-WAP49, an immunodominant antigen secreted by adult Trichuris stichocytes into the mucosa of the cecum to which the parasite attaches, is under development. The prototype is being evaluated in a mouse model of Trichuris muris infection, with the ultimate goal of producing a mucosal vaccine through intranasal delivery. Intranasal immunization of mice with Tm-WAP49 formulated with the adjuvant OCH, a truncated analog of alpha-GalCer with adjuvanticity to stimulate natural killer T cells (NKT) and mucosal immunity, induced significantly high levels of IgG and its subclasses (IgG1 and IgG2a) in immunized mice. This also resulted in a significant reduction of worm burden after challenge with T. muris-infective eggs. The addition of QS-21 adjuvant to this vaccine formulation further reduced worm counts. The improved protection from the dual-adjuvanted vaccine correlated with higher serum antibody responses (IgG, IgG1, IgG2a, IgA) as well as with the induction of antigen-specific IgA in the nasal mucosa. It was also associated with the robust cellular responses including functional subsets of CD4 T cells producing IL-4, and cytotoxic CD8 T cells expressing granzyme B. The worm reduction achieved by mucosal immunization was higher than that induced by subcutaneous immunization. Intranasal immunization also induced a significantly higher nasal mucosa-secreted antigen-specific IgA response, as well as higher functional cellular responses including CD4+IL4+ (Th1) and CD8+GnzB+ (Th2) T cells, and antigen-specific INFγ-producing T cells in both spleen and MLNs and antibody-producing B cells (CD19+B220+/B220+GL7+). Mucosal immunization further induced long-term T lymphocyte memory with increased central (CD62L+CD44+) and effector (CD62L-CD44+) memory subsets of both CD4 and CD8 T cells at 60 days after the last immunization. In summary, intranasal immunization with recombinant Tm-WAP49 protein induced strong protection versus murine trichuriasis. It represents a promising vaccination approach against intestinal nematodes.

ASCVac-1, a Multi-Peptide Chimeric Vaccine, Protects Mice Against Ascaris suum Infection

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

Oral immunization with attenuated Salmonella encoding an elastase elicits protective immunity against Trichinella spiralis infection

Elastase belongs to the serine protease family. Previous studies showed that Trichinella spiralis elastase (TsE) was highly expressed in intestinal infective larvae (IIL). Recombinant TsE (rTsE) promoted the larval intrusion of enteral epithelium cells (IECs), whereas anti-rTsE antibodies and siRNA impeded larval intrusion. Subcutaneous vaccination of mice with rTsE showed a partial protective immunity, suggesting that TsE might be a promising vaccine target against Trichinella infection. In this study, complete TsE cDNA sequence was cloned into pcDNA3.1, and the rTsE DNA was transformed into attenuated S. typhimurium strain ΔcyaSL1344. Oral vaccination of mice with TsE DNA elicited a systemic Th1/Th2/Treg mixed immune response and gut local mucosal sIgA response. Immunized mice exhibited a significant immune protection against T. spiralis larval challenge, as demonstrated by a 52.48% reduction of enteral adult worms and a 69.43% reduction of muscle larvae. The protection might be related to the TsE-induced production of intestinal mucus, specific anti-TsE sIgA and IgG, and secretion of IFN-γ, IL-2, IL-4 and IL-10, which protected gut mucosa from larval intrusion, suppressed worm development and impeded female reproduction. The results demonstrated that attenuated Salmonella-delivered TsE DNA vaccine provided a prospective strategy for the control of Trichinella infection in food animals.

Anti-pinworm activity of novel coumarin-based trisubstituted methanes in Syphacia obvelata-infected mice.

The control of pinworms mainly relies on use of anthelmintic drugs. At present, there exists only few medications against pinworms, and their repeated use pose a serious risk of resistance development. Therefore, new anti-pinworm drugs are required to overcome the risk of resistance. This study reports the anti-pinworm activity of three novel coumarin-based trisubstituted methanes (TRSMs), i.e., 6-Amino-5-((4-hydroxy-2-oxo-2H-chromen-3-yl)(4-fluoro-phenyl)methyl)-1,3-dimethyl-pyrimidine-2,4(1H,3H)-dione (1), 6-Amino-5-((4-hydroxy-2-oxo-2H-chromen-3-yl)(4-chlor-ophenyl)methyl)-1,3-dimethyl-pyrimidine-2,4(1H,3H)-dione (2) and 6-Amino-5-((4-hydroxy-2-oxo-2H-chromen-3-yl)(4-bromophenyl)methyl)-1,3-dimethyl-pyrimidine-2,4(1H,3H)-dione (3) in Syphacia obvelata-infected mice. The oral acute toxicity of compounds was examined using the OECD guidelines. The findings of this study reveal that TRSM analogues 1 and 2, at a single 80mg/kg dose given for 5days, can reduce about 90% of pinworm worm burden in mice, compared to 98% worm reduction shown by 20mg/kg dose of albendazole, the reference drug, on the 12day of infection. In particular, the fluoro-and bromo-substituents in the phenyl ring of synthesized derivatives greatly influence the efficacy of candidates. The oral acute toxicity of TRSMs was observed to be greater than 2000mg/kg body weight for mice. Taken together, our study suggests that studied novel coumarin-based trisubstituted methanes could serve as suitable candidates for the development of new anti-pinworm drugs.

Oral vaccination with recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase elicited a protective immunity in BALB/c mice.

BackgroundTrichinellosis is a serious zoonotic disease distributed around the world. It is needed to develop a safe, effective and feasible anti-Trichinella vaccine for prevention and control of trichinellosis. The aim of this study was to construct a recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase (TsPPase) and investigate its immune protective effects against T. spiralis infection.Methodology/Principal findingsThe growth of recombinant L. plantarum was not affected by TsPPase/pSIP409-pgsA′ plasmid, and the recombinant plasmid was inherited stably in bacteria. Western blot and immunofluorescence assay (IFA) indicated that the rTsPPase was expressed on the surface of recombinant L. plantarum. Oral vaccination with rTsPPase induced higher levels of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA) in BALB/c mice. ELISA analysis revealed that the levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer’s patches were evidently increased at 2–4 weeks following vaccination, compared to MRS (De Man, Rogosa, Sharpe) medium control group (P < 0.05). Immunization of mice with rTsPPase exhibited a 67.18, 54.78 and 51.91% reduction of intestinal infective larvae, adult worms and muscle larvae at 24 hours post infection (hpi), 6 days post infection (dpi) and 35 dpi, respectively (P < 0.05), and the larval molting and development was significantly inhibited by 45.45% at 24 hpi, compared to the MRS group.ConclusionsTsPPase plays a crucial role in T. spiralis molting and development, oral vaccination with rTsPPase induced a significant local mucosal sIgA response and systemic Th1/Th2 immune response, and immune protection against T. spiralis infection in BALB/c mice.

The Role of Citrate Transporter INDY in Metabolism and Stem Cell Homeostasis.

I’m Not Dead Yet (Indy) is a fly gene that encodes a homologue of mammalian SLC13A5 plasma membrane citrate transporter. Reducing expression of Indy gene in flies, and its homologues in worms, extends longevity. Indy reduction in flies, worms, mice and rats affects metabolism by regulating the levels of cytoplasmic citrate, inducing a state similar to calorie restriction. Changes include lower lipid levels, increased insulin sensitivity, increased mitochondrial biogenesis, and prevention of weight gain, among others. The INDY protein is predominantly expressed in fly metabolic tissues: the midgut, fat body and oenocytes. Changes in fly midgut metabolism associated with reduced Indy gene activity lead to preserved mitochondrial function and reduced production of reactive oxygen species. All these changes lead to preserved intestinal stem cell homeostasis, which has a key role in maintaining intestinal epithelium function and enhancing fly healthspan and lifespan. Indy gene expression levels change in response to caloric content of the diet, inflammation and aging, suggesting that INDY regulates metabolic adaptation to nutrition or energetic requirements by controlling citrate levels.