Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species.

BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.

A novel mRNA vaccine, TGGT1_278620 mRNA-LNP, prolongs the survival time in BALB/c mice with acute toxoplasmosis.

IMPORTANCE: Toxoplasma gondii, an obligate intracellular eukaryotic parasite, can infect about one-third of the world's population. One vaccine, Toxovax, has been developed and licensed commercially; however, it is only used in the sheep industry to reduce the losses caused by congenital toxoplasmosis. Various other vaccine approaches have been explored, including excretory secretion antigen vaccines, subunit vaccines, epitope vaccines, and DNA vaccines. However, current research has not yet developed a safe and effective vaccine for T. gondii. Here, we generated an mRNA vaccine candidate against T. gondii. We investigated the efficacy of vaccination with a novel identified candidate, TGGT1_278620, in a mouse infection model. We screened T. gondii-derived protective antigens at the genome-wide level, combined them with mRNA-lipid nanoparticle vaccine technology against T. gondii, and investigated immune-related factors and mechanisms. Our findings might contribute to developing vaccines for immunizing humans and animals against T. gondii.

Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats.

BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.

Mining host candidate regulators of schistosomiasis-induced liver fibrosis in response to artesunate therapy through transcriptomics approach.

BACKGROUND: Artesunate (ART) has been reported to have an antifibrotic effect in various organs. The underlying mechanism has not been systematically elucidated. We aimed to clarify the effect of ART on liver fibrosis induced by Schistosoma japonicum (S. japonicum) in an experimentally infected rodent model and the potential underlying mechanisms. METHODS: The effect of ART on hepatic stellate cells (HSCs) was assessed using CCK-8 and Annexin V-FITC/PI staining assays. The experimental model of liver fibrosis was established in the Mongolian gerbil model infected with S. japonicum cercariae and then treated with 20 mg/kg or 40 mg/kg ART. The hydroxyproline (Hyp) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in liver tissue were measured and histopathological changes of liver tissues were observed. Whole-transcriptome RNA sequencing (RNA-seq) of the liver tissues was performed. Differentially expressed genes (DEGs) were identified using bioinformatic analysis and verified by quantitative PCR (qPCR) and western blot assay. RESULTS: ART significantly inhibited the proliferation and induce the apoptosis of HSCs in a dose-dependent manner. In vivo, Hyp content decreased significantly in the ART-H group compared to the model (MOD) group and GPX activity was significantly higher in the ART-H group than in the MOD group. Besides, ART treatment significantly reduced collagen production (p <0.05). A total of 158 DEGs and 44 differentially expressed miRNAs related to ART-induced anti-schistosomiasis liver fibrosis were identified. The qPCR and western blot results of selected DEGs were consistent with the sequencing results. These DEGs were implicated in key pathways such as immune and inflammatory response, integrin-mediated signaling and toll-like receptor signaling pathways. CONCLUSION: ART is effective against liver fibrosis using Mongolian gerbil model induced by S. japonicum infection. We identified host candidate regulators of schistosomiasis-induced liver fibrosis in response to ART through transcriptomics approach.

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats.

BACKGROUND: Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats. METHODS: First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel-Cox) test, Student's t test and one-way ANOVA. RESULTS: The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8+ T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%). CONCLUSIONS: The avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.

Immunization with a novel mRNA vaccine, TGGT1_216200 mRNA-LNP, prolongs survival time in BALB/c mice against acute toxoplasmosis.

Toxoplasma gondii, a specialized intracellular parasite, causes a widespread zoonotic disease and is a severe threat to social and economic development. There is a lack of effective drugs and vaccines against T. gondii infection. Recently, mRNA vaccines have been rapidly developed, and their packaging materials and technologies are well established. In this study, TGGT1_216200 (TG_200), a novel molecule from T. gondii, was identified using bioinformatic screening analysis. TG_200 was purified and encapsulated with a lipid nanoparticle (LNP) to produce the TG_200 mRNA-LNP vaccine. The immune protection provided by the new vaccine and its mechanisms after immunizing BABL/C mice via intramuscular injection were investigated. There was a strong immune response when mice were vaccinated with TG_200 mRNA-LNP. Elevated levels of anti-T. gondii-specific immunoglobulin G (IgG), and a higher IgG2a-to-IgG1 ratio was observed. The levels of interleukin-12 (IL-12), interferon-γ (IFN-γ), IL-4, and IL-10 were also elevated. The result showed that the vaccine induced a mixture of Th1 and Th2 cells, and Th1-dominated humoral immune response. Significantly increased antigen-specific splenocyte proliferation was induced by TG_200 mRNA-LNP immunization. The vaccine could also induce T. gondii-specific cytotoxic T lymphocytes (CTLs). The expression levels of interferon regulatory factor 8 (IRF8), T-Box 21 (T-bet), and nuclear factor kappa B (NF-κB) were significantly elevated after TG_200 mRNA-LNP immunization. The levels of CD83, CD86, MHC-I, MHC-II, CD8, and CD4 molecules were also higher. The results indicated that TG_200 mRNA-LNP produced specific cellular and humoral immune responses. Most importantly, TG_200 mRNA-LNP immunized mice survived significantly longer (19.27 ± 3.438 days) than the control mice, which died within eight days after T. gondii challenge (P< 0.001). The protective effect of adoptive transfer was also assessed, and mice receiving serum and splenocytes from mice immunized with TG_200 mRNA-LNP showed improved survival rates of 9.70 ± 1.64 days and, 13.40 ± 2.32 days, respectively (P< 0.001). The results suggested that TG_200 mRNA-LNP is a safe and promising vaccine against T. gondii infection.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China.

BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

Toxoplasmosis vaccines: what we have and where to go?

Despite recent major advances in developing effective vaccines against toxoplasmosis, finding new protective vaccination strategies remains a challenging and elusive goal as it is critical to prevent the disease. Over the past few years, various experimental approaches have shown that developing an effective vaccine against T. gondii is achievable. However, more remains unknown due to its complicated life cycle, difficulties in clinical translation, and lack of a standardized platform. This minireview summarizes the recent advances in the development of T. gondii vaccines and the main obstacles to developing a safe, effective and durable T. gondii vaccine. The successes and failures in developing and testing vaccine candidates for the T. gondii vaccine are also discussed, which may facilitate the future development of T. gondii vaccines.

Anti-Tumor Effect of Parasitic Protozoans.

The immune system may aberrantly silence when against "altered self", which consequently may develop into malignancies. With the development of tumor immunology and molecular biology, the deepened understanding of the relationship between parasites and tumors shifts the attitude towards parasitic pathogens from elimination to utilization. In recent years, the antitumor impact implemented by protozoan parasites and the derived products has been confirmed. The immune system is activated and enhanced by some protozoan parasites, thereby inhibiting tumor growth, angiogenesis, and metastasis in many animal models. In this work, we reviewed the available information on the antitumor effect of parasitic infection or induced by parasitic antigen, as well as the involved immune mechanisms that modulate cancer progression. Despite the fact that clinical trials of the protozoan parasites against tumors are limited and the specific mechanisms of the effect on tumors are not totally clear, the use of genetically modified protozoan parasites and derived molecules combined with chemotherapy could be an important element for promoting antitumor treatment in the future.

Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis.

Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)|-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.

Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

The coronavirus disease 2019 (COVID-19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen.

A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.

TITLE: Un nouveau dispositif de bandelette à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) pour la détection rapide de Toxoplasma gondii dans le sang des chats et chiens errants. ABSTRACT: Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire qui provoque la toxoplasmose et menace la santé humaine et les animaux à sang chaud dans le monde entier. Des méthodes de diagnostic simples et applicables sont nécessaires de toute urgence pour guider le développement d'approches efficaces pour la prévention de la toxoplasmose. La plupart des outils de diagnostic moléculaire pour l'infection par T. gondii nécessitent des compétences techniques élevées, un équipement sophistiqué et un environnement de laboratoire contrôlé. Dans cette étude, nous avons développé un test par bandelettes à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) qui cible spécifiquement les 529 pb qui détectent une infection par T. gondii. Ce nouvel appareil portable est universel, rapide, convivial et garantit une sensibilité expérimentale ainsi qu'un faible risque de contamination par aérosol. Notre test LAMP-LFD a une limite de détection de 1 fg d'ADN de T. gondii et ne montre aucune réaction croisée avec d'autres pathogènes parasites, y compris Cryptosporidium parvum, Leishmania donovani et Plasmodium vivax. Nous avons validé le test en détectant T. gondii dans l'ADN extrait d'échantillons de sang prélevés sur 318 chats et chiens errants prélevés dans les villes de Deqing, Wenzhou, Yiwu, Lishui et Zhoushan dans la province du Zhejiang, dans l'est de la Chine. Le dispositif LAMP-LFD a détecté la prévalence de l'ADN de T. gondii chez respectivement 4,76 et 4,69% des chats et chiens errants. En conclusion, le test LAMP-LFD développé est efficace, minimise la contamination par les aérosols et convient donc à la détection de T. gondii dans les établissements médicaux simples et sur le terrain.

Predictive Analysis of the Neutralization Activity in Convalescent Plasmas From COVID-19 Recovered Patients in Zhejiang Province, China, January-March, 2020.

Background: Convalescent plasma (CP) transfusion is considered to be the priority therapeutic option for COVID-19 inpatients when no specific drugs are available for emerging infections. An alternative, simple, and sensitive method is urgently needed for clinical use to detect neutralization activity of the CP to avoid the use of inconvenient micro-neutralization assay. Method: This study aims to explore optimal index in predicting the COVID-19 CP neutralization activity (neutralizing antibody titers, NAb titers) in an indirect ELISA format. Fifty-seven COVID-19-recovered patients plasma samples were subjected to anti-SARS-CoV-2 RBD, S1, and N protein IgG antibody by indirect ELISA. Results: ELISA-RBD exhibited high specificity (96.2%) and ELISA-N had high sensitivity (100%); while ELISA-S1 had low sensitivity (86.0%) and specificity (73.1%). Furthermore, ELISA-RBD IgG titers and pseudovirus-based NAb titers correlated significantly, with R2 of 0.2564 (P < 0.0001). Conclusion: ELISA-RBD could be a substitute for the neutralization assay in resource-limited situations to screen potential plasma donors for further plasma infusion therapy.

Early Detection of Trichinella spiralis DNA in Rat Feces Based on Tracing Phosphate Ions Generated During Loop-Mediated Isothermal Amplification.

Early diagnosis of trichinellosis is still difficult because of the lack of specific symptoms and limited window for serological detection. Here we established an assay based on tracing phosphate ions generated during loop-mediated isothermal amplification (LAMP) to detect Trichinella spiralis DNA in rat feces during its early stage of infection. By targeting a 1.6-kb repetitive element of Tri. spiralis, the assay was able to detect Tri. spiralis DNA in the feces of all infected rats as early as 1 day postinfection (dpi). The positive detection lasted to 7 dpi in the rats infected with 250 muscle larvae, and 21 dpi in the rats infected with 5,000 larvae. The assay was highly sensitive, and could detect 1.7 femtograms (fg) of Tri. spiralis DNA with high specificity, and with no cross reactivity with the DNA from Anisakis pegreffii, Gnathostoma spinigerum, Angiostrongylus cantonensis, Enterobius vermicularis, Schistosoma japonicum, and Trypanosoma evansi. Our present study provided a reliable technique for the early diagnosis of trichinellosis with the advantages of simplicity and speed, as well as high sensitivity and specificity.

A Carbamoyl Phosphate Synthetase II (CPSII) Deletion Mutant of Toxoplasma gondii Induces Partial Protective Immunity in Mice.

Toxoplasma gondii is an obligate intracellular protozoan parasite. T. gondii primarily infection in pregnant women may result in fetal abortion, and infection in immunosuppressed population may result in toxoplasmosis. Carbamoyl phosphate synthetase II (CPSII) is a key enzyme in the de novo pyrimidine-biosynthesis pathway, and has a crucial role in parasite replication. We generated a mutant with complete deletion of CPSII via clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 in type-1 RH strain of T. gondii. We tested the intracellular proliferation of this mutant and found that it showed significantly reduced replication in vitro, though CPSII deletion did not completely stop the parasite growth. The immune responses induced by the infection of RHΔCPSII tachyzoites in mice were evaluated. During infection in mice, the RHΔCPSII mutant displayed notable defects in replication and virulence, and significantly enhanced the survival of mice compared with survival of RH-infected mice. We tracked parasite propagation from ascitic fluid in mice infected with the RHΔCPSII mutant, and few tachyzoites were observed at early infection. We also observed that the RHΔCPSII mutant induced greater accumulation of neutrophils. The mutant induced a higher level of T-helper type-1 cytokines [interferon (IFN)-γ, interleukin (IL)-12]. The mRNA levels of signal transducer and activator of transcription cellular transcription factor 1 and IFN regulatory factor 8 were significantly higher in the RHΔCPSII mutant-infected group. Together, these data suggest that CPSII is crucial for parasite growth, and that strains lack the de novo pyrimidine biosynthesis pathway and salvage pathway may become a promising live attenuated vaccine to prevent infection with T. gondii.

The Virulence-Related MYR1 Protein of Toxoplasma gondii as a Novel DNA Vaccine Against Toxoplasmosis in Mice.

Toxoplasma gondii causes serious public health problems, but there is no effective treatment strategy against it currently. DNA vaccines have shown promising findings in this regard. MYR1 is a new virulence factor identified in T. gondii that may have potential as a DNA vaccine candidate. We constructed a recombinant eukaryotic plasmid, pVAX1-MYR1, as a DNA vaccine, injected it intramuscularly into BALB/c mice, and evaluated its immunoprotective effects. pVAX1-MYR1 immunization induced a sequential Th1 and Th2 T-cell response, as indicated by high levels of Th1 and mixed Th1/Th2 cytokines at 2 and 6 weeks after immunization, respectively. These findings were corroborated by the antibody assays too. In addition, increased levels of antigen-specific lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, cytotoxic T lymphocyte activity and cytokine (IFN-γ, IL-12, and IL-10) production were also observed in the immunized mice. These findings showed that pVAX1-MYR1 stimulated humoral and cellular immune responses in the immunized mice. The increased production of IFN-γ and IL-12 was correlated with increased expression of the T-bet and p65 genes of the NF-κB pathway. However, no significant increase was observed in the level of IL-4. The survival of mice immunized with pVAX1-MYR1 was also significantly prolonged compared with the control group mice. Based on all the above findings, the current study proposes that pVAX1-MYR1 can induce a T. gondii-specific immune response and should therefore be considered as a promising vaccine candidate against toxoplasmosis. To the best of our knowledge, this is the first report to evaluate the immunoprotective value of an MYR1-based DNA vaccine against T. gondii.

AKT2 phosphorylation of hexokinase 2 at T473 promotes tumorigenesis and metastasis in colon cancer cells via NF-κB, HIF1α, MMP2, and MMP9 upregulation.

It has been well-established that AKT2 plays an important role in the development and progression of colon cancer; however, its precise function remains unclear. In the present study, we found that AKT2 can interact with and phosphorylate hexokinase 2 (HK2), the rate-limiting enzyme in glycolysis. Moreover, threonine phosphorylation dramatically increases its catalytic activity and enhances glycolysis. Mechanistically, AKT2 phosphorylation of HK2 at T473 was found to increase hexokinase activity and lactic acid production. A mutation in the AKT2 phosphorylation site of HK2 substantially reduced the stimulating effects of AKT2 on glycolysis, cellular apoptosis, invasion, tumorigenesis, and metastasis. In addition, AKT2 regulated NF-κB, HIF1Α, MMP2, and MMP9 via the phosphorylation of HK2 at the T473 site. Taken together, AKT2 increases the invasion, tumorigenesis, and metastasis of colon cancer cells in vitro and promotes lung metastasis in nude mice in vivo through the phosphorylation of the T473 site of HK2 by upregulating NF-κB, HIF1α, MMP2, and MMP9. In conclusion, our findings highlight a novel mechanism for the AKT2-HK2-NF-κB/HIF1α/MMP2/MMP9 axis in the regulation of colon cancer progression. Moreover, our results suggest that both AKT2 and HK2 may be potential targets for the treatment of colon cancer.

GRA24-Based DNA Vaccine Prolongs Survival in Mice Challenged With a Virulent Toxoplasma gondii Strain.

Toxoplasma gondii causes infections in a wide range of intermediate hosts and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Dense granule protein 24 (GRA24) is a novel essential virulence factor that is transferred into the nucleus of host cells from the parasitophorous vacuole to regulate gene expression. In the present study, bioinformatic analysis showed that GRA24 had a high score for B-cell and T-cell epitopes compared with surface antigen 1 (SAG1), which has been studied as a promising vaccine candidate. As a DNA vaccine, pVAX1-GRA24 was injected intramuscularly into BALB/c mice and the induced immune response was evaluated. pVAX1-GRA24 induced high levels of a mixed Th1/Th2 cytokines at 6 weeks after immunization. Antibody determinations, cytokines [interferon gamma (IFN-γ), interleukin (IL)-12, IL-4, IL-10], antigen-specific lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, and cytotoxic T lymphocyte activity showed that mice immunized with pVAX1-GRA24 produced specific humoral and cellular immune responses. The expression levels of interferon regulatory factor 8 (IRF8), nuclear factor kappa B (NF-κB), and T-Box 21 (T-bet) were significantly higher in the pVAX1-GRA24 immunization group than in the control groups. Survival times were prolonged significantly (24.6 ± 5.5 days) in the mice immunized with pVAX1-GRA24 compared with the mice in the control groups, which died within 7 days of T. gondii challenge (p < 0.05). The results of the present study showed that pVAX1-GRA24 induced a T. gondii-specific immune response and thus represents a promising candidate vaccine to treat toxoplasmosis.

Early Detection of Toxoplasma gondii Infection In Mongolian Gerbil By Quantitative Real-Time PCR.

Toxoplasmosis, caused by Toxoplasma gondii, is associated with several clinical syndromes, including encephalitis, chorioretinitis, and congenital infection. Toxoplasma gondii is a ubiquitous apicomplexan parasite found in both humans and animals. Mongolian gerbils, which are more susceptible to both high- and low-virulence Toxoplasma strains compared with mice, are considered useful models for assessing diagnosis and treatment methods for toxoplasmosis, as well as infection by and host defense to this organism. Here we established a quantitative real-time polymerase chain reaction (qPCR) method targeting the B1 gene for early and specific detection of T. gondii infection in Mongolian gerbil. The detection limit of the developed qPCR was approximately 1 T. gondii tachyzoite. This method was also applied to detect T. gondii genomic DNA in experimentally infected Mongolian gerbils, with positive results in blood (66.7%), liver (73.3%), lung (80.0%), spleen (80.0%), and peritoneal fluid (66.7%) samples as early as 1 day postinfection. Specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Cryptosporidium parvum, Eimeria tenella, Trypanosoma evansi, Schistosoma japonicum, Angiostrongylus cantonensis, and Strongyloides stercoralis. This study first reports the use of Mongolian gerbils as an animal model for early diagnosis of toxoplasmosis by qPCR.