A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

The volatilome signatures of Plasmodium falciparum parasites during the intraerythrocytic development cycle in vitro under exposure to artemisinin drug.

Volatile organic compounds (VOCs) comprise a diverse range of metabolites with high vapour pressure and low boiling points. Although they have received attention, they are a largely unexplored part of the metabolome. Previous studies have shown that malaria infections produce characteristic, definitive, and detectable volatile signatures. Many transcriptional and metabolic differences are observed at different stages of the parasite Intraerythrocytic Developmental Cycle (IDC) as well as when artemisinin-resistant parasites are put under drug pressure. This prompted our research to characterize whether these responses are reflected at a volatile level in malaria during the IDC stages using gas chromatography-mass spectrometry. We investigated whether the resistant P. falciparum parasites would produce their own characteristic volatilome profile compared to near-isogenic wild-type parasite in vitro; firstly at three different stages of the IDC and secondly in the presence or absence of artemisinin drug treatment. Finally, we explored the VOC profiles from two media environments (Human serum and Albumax) of recently lab-adapted field parasite isolates, from Southeast Asia and West/East Africa, compared to long-term lab-adapted parasites. Recognizable differences were observed between IDC stages, with schizonts having the largest difference between wild type and resistant parasites, and with cyclohexanol and 2,5,5-trimethylheptane only present for resistant schizonts. Artemisinin treatment had little effect on the resistant parasite VOC profile, whilst for the wild type parasites compounds ethylbenzene and nonanal were greatly affected. Lastly, differing culturing conditions had an observable impact on parasite VOC profile and clustering patterns of parasites were specific to geographic origin. The results presented here provide the foundation for future studies on VOC based characterization of P. falciparum strains differing in abilities to tolerate artemisinin.

Multi-omics dissection of stage-specific artemisinin tolerance mechanisms in Kelch13-mutant Plasmodium falciparum.

AIMS: We investigated the stage-specific mechanisms of partial resistance to artemisinin (ART, an antimalarial drug) in Plasmodium falciparum (P. falciparum) carrying the Kelch13 C580Y mutation. METHODS: Using fluorescence labeling and activity-based protein profiling, we systematically profile the ART activation levels in P. falciparum during the entire intra-erythrocytic developmental cycle (IDC), and determined the ART-targets profile of the ART-sensitive and -resistant strains at different stages. We retrieved and integrated datasets of single-cell transcriptomics and label-free proteomics across three IDC stages of wild-type P. falciparum. We also employed lipidomics to validate lipid metabolic reprogramming in the resistant strain. RESULTS: The activation and expression patterns of genes and proteins of ART-targets in both ART-sensitive and resistant strains varied at different stages and periods of P. falciparum development, with the late trophozoite stage harboring the largest number of ART targets. We identified and validated 36 overlapping targets, such as GAPDH, EGF-1a, and SpdSyn, during the IDC stages in both strains. We revealed the ART-insensitivity of fatty acid-associated activities in the partially resistant strain at both the early ring and early trophozoite stages. CONCLUSIONS: Our multi-omics strategies provide novel insights into the mechanisms of ART partial resistance in Kelch13 mutant P. falciparum, demonstrating the stage-specific interaction between ART and malaria parasites.

A review of malaria molecular markers for drug resistance in Plasmodium falciparum and Plasmodium vivax in China.

China has now achieved the elimination of malaria, but it still faces severe challenges in the post-elimination stage. China continues to be plagued by imported malaria cases, and preventing re-transmission of imported malaria is critical. The effectiveness of antimalarial drugs for malaria control largely depends on the study of drug resistance markers in vitro. Monitoring molecular markers of parasite-associated drug resistance can help predict and manage drug resistance. There is currently a lack of systematic reviews of molecular markers for indigenous and imported malaria in China. Therefore, this review summarizes the published articles related to molecular marker polymorphism of indigenous and imported malaria cases in China in the past two decades, to study the mutation frequency and distribution of crt, mdr1, dhps, dhfr and K13 gene resistance-related loci. This can provide a whole picture of molecular markers and the resistance mutations of imported cases in China, which has certain significance for drug resistance surveillance planning, safe and effective treatment, and preventing the recurrence of local transmission by imported malaria in China in the future.

Naturally Acquired Kelch13 Mutations in Plasmodium falciparum Strains Modulate In Vitro Ring-Stage Artemisinin-Based Drug Tolerance and Parasite Survival in Response to Hyperoxia.

The ring-stage survival assay was utilized to assess the impact of physiological hyperoxic stress on dihydroartemisinin (DHA) tolerance for a panel of Plasmodium falciparum strains with and without Kelch13 mutations. Strains without naturally acquired Kelch13 mutations or the postulated genetic background associated with delayed parasite clearance time demonstrated reduced proliferation under hyperoxic conditions in the subsequent proliferation cycle. Dihydroartemisinin tolerance in three isolates with naturally acquired Kelch13 mutations but not two genetically manipulated laboratory strains was modulated by in vitro hyperoxic stress exposure of early-ring-stage parasites in the cycle before drug exposure. Reduced parasite tolerance to additional derivatives, including artemisinin, artesunate, and OZ277, was observed within the second proliferation cycle. OZ439 and epoxomicin completely prevented parasite survival under both hyperoxia and normoxic in vitro culture conditions, highlighting the unique relationship between DHA tolerance and Kelch13 mutation-associated genetic background. IMPORTANCE Artemisinin-based combination therapy (ACT) for treating malaria is under intense scrutiny following treatment failures in the Greater Mekong subregion of Asia. This is further compounded by the potential for extensive loss of life if treatment failures extend to the African continent. Although Plasmodium falciparum has become resistant to all antimalarial drugs, artemisinin "resistance" does not present in the same way as resistance to other antimalarial drugs. Instead, a partial resistance or tolerance is demonstrated, associated with the parasite's genetic profile and linked to a molecular marker referred to as K13. It is suggested that parasites may have adapted to drug treatment, as well as the presence of underlying population health issues such as hemoglobinopathies, and/or environmental pressures, resulting in parasite tolerance to ACT. Understanding parasite evolution and control of artemisinin tolerance will provide innovative approaches to mitigate the development of artemisinin tolerance and thereby artemisinin-based drug treatment failure and loss of life globally to malaria infections.

Molecular Surveillance of Artemisinin-Based Combination Therapies Resistance in Plasmodium falciparum Parasites from Bioko Island, Equatorial Guinea.

Artemisinin-based combination therapies (ACTs) resistance has emerged and could be diffusing in Africa. As an offshore island on the African continent, the island of Bioko in Equatorial Guinea is considered severely affected and resistant to drug-resistant Plasmodium falciparum malaria. However, the spatial and temporal distribution remain unclear. Molecular monitoring targeting the Pfcrt, Pfk13, Pfpm2, and Pfmdr1 genes was conducted to provide insight into the impact of current antimalarial drug resistance on the island. Furthermore, polymorphic characteristics, haplotype network, and the effect of natural selection of the Pfk13 gene were evaluated. A total of 152 Plasmodium falciparum samples (collected from 2017 to 2019) were analyzed for copy number variation of the Pfpm2 gene and Pfk13, Pfcrt, and Pfmdr1 mutations. Statistical analysis of Pfk13 sequences was performed following different evolutionary models using 96 Bioko sequences and 1322 global sequences. The results showed that the prevalence of Pfk13, Pfcrt, and Pfmdr1 mutations was 73.68%, 78.29%, and 75.66%, respectively. Large proportions of isolates with multiple copies of Pfpm2 were observed (67.86%). In Bioko parasites, the genetic diversity of Pfk13 was low, and purifying selection was suggested by Tajima's D test (-1.644, P > 0.05) and the dN/dS test (-0.0004438, P > 0.05). The extended haplotype homozygosity analysis revealed that Pfk13_K189T, although most frequent in Africa, has not yet conferred a selective advantage for parasitic survival. The results suggested that the implementation of continuous drug monitoring on Bioko Island is an essential measure. IMPORTANCE Malaria, one of the tropical parasitic diseases with a high transmission rate in Bioko Island, Equatorial Guinea, especially caused by P. falciparum is highly prevalent in this region and is commonly treated locally with ACTs. The declining antimalarial susceptibility of artemisinin-based drugs suggested that resistance to artemisinin and its derivatives is developing in P. falciparum. Copy number variants in Pfpm2 and genetic polymorphisms in Pfk13, Pfcrt, and Pfmdr1 can be used as risk assessment indicators to track the development and spread of drug resistance. This study reported for the first time the molecular surveillance of Pfpm2, Pfcrt, Pfk13, and Pfmdr1 genes in Bioko Island from 2017 to 2019 to assess the possible risk of local drug-resistant P. falciparum.

Novel pfk13 polymorphisms in Plasmodium falciparum population in Ghana.

The molecular determinants of Plasmodium falciparum artemisinin resistance are the single nucleotide polymorphisms in the parasite's kelch propeller domain, pfk13. Validated and candidate markers are under surveillance in malaria endemic countries using artemisinin-based combination therapy. However, pfk13 mutations which may confer parasite artemisinin resistance in Africa remains elusive. It has therefore become imperative to report all observed pfk13 gene polymorphisms in malaria therapeutic efficacy studies for functional characterization. We herein report all novel pfk13 mutations observed only in the Ghanaian parasite population. In all, 977 archived samples from children aged 12 years and below with uncomplicated malaria from 2007 to 2017 were used. PCR/Sanger sequencing analysis revealed 78% (763/977) of the samples analyzed were wild type (WT) for pfk13 gene. Of the 214 (22%) mutants, 78 were novel mutations observed only in Ghana. The novel SNPs include R404G, P413H, N458D/H/I, C473W/S, R529I, M579T/Y, C580R/V, D584L, N585H/I, Q661G/L. Some of the mutations were sites and ecological zones specific. There was low nucleotide diversity and purifying selection at the pfk13 locus in Ghanaian parasite population. With increasing drug pressure and its consequent parasite resistance, documenting these mutations as baseline data is crucial for future molecular surveillance of P. falciparum resistance to artemisinin in Ghana.

Novel Murine Pancreatic Tumor Model Demonstrates Immunotherapeutic Control of Tumor Progression by a Toxoplasma gondii Protein.

Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer-related death in the United States, with few effective treatments available and only 10% of those diagnosed surviving 5 years. Although immunotherapeutics is a growing field of study in cancer biology, there has been little progress in its use for the treatment of pancreatic cancer. Pancreatic cancer is considered a nonimmunogenic tumor because the tumor microenvironment does not easily allow for the immune system, even when stimulated, to attack the cancer. Infection with the protozoan parasite Toxoplasma gondii has been shown to enhance the immune response to clear cancer tumors. A subset of T. gondii proteins called soluble Toxoplasma antigen (STAg) contains an immunodominant protein called profilin. Both STAg and profilin have been shown to stimulate an immune response that reduces viral, bacterial, and parasitic burdens. Here, we use STAg and profilin to treat pancreatic cancer in a KPC mouse-derived allograft murine model. These mice exhibit pancreatic cancer with both Kras and P53 mutations as subcutaneous tumors. Pancreatic cancer tumors in C57BL/6J mice with a wild-type background showed a significant response to treatment with either profilin or STAg, exhibiting a decrease in tumor volume accompanied by an influx of CD4+ and CD8+ T cells into the tumors. Both IFN-γ-/- mice and Batf3-/- mice, which lack conventional dendritic cells, failed to show significant decreases in tumor volumes when treated. These results indicate that gamma interferon (IFN-γ) and dendritic cells may play critical roles in the immune response necessary to treat pancreatic cancer.

Identification and Immune Assessment of T Cell Epitopes in Five Plasmodium falciparum Blood Stage Antigens to Facilitate Vaccine Candidate Selection and Optimization.

The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested Plasmodium falciparum (Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using in silico, in vitro, and ex vivo methodologies. We employed in silico T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding in vitro, to verify the in silico predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles in vitro. The overall epitope prediction to in vitro HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-γ T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses - by percentage of responders and response magnitude - associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between in silico epitope predictions and ex vivo IFN-γ recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by in vitro HLA binding validation for all five proteins and ex vivo T cell response confirmation for RH5.

Injection with Toxoplasma gondii protein affects neuron health and survival.

Toxoplasma gondii is an intracellular parasite that causes a long-term latent infection of neurons. Using a custom MATLAB-based mapping program in combination with a mouse model that allows us to permanently mark neurons injected with parasite proteins, we found that Toxoplasma-injected neurons (TINs) are heterogeneously distributed in the brain, primarily localizing to the cortex followed by the striatum. In addition, we determined that cortical TINs are commonly (>50%) excitatory neurons (FoxP2+) and that striatal TINs are often (>65%) medium spiny neurons (MSNs) (FoxP2+). By performing single neuron patch clamping on striatal TINs and neighboring uninfected MSNs, we discovered that TINs have highly aberrant electrophysiology. As approximately 90% of TINs will die by 8 weeks post-infection, this abnormal physiology suggests that injection with Toxoplasma protein-either directly or indirectly-affects neuronal health and survival. Collectively, these data offer the first insights into which neurons interact with Toxoplasma and how these interactions alter neuron physiology in vivo.

Toxoplasma gondii is an intracellular parasite that infects the brain. Whereas most microbial infections of the brain result in severe illness or death, Toxoplasma gondii infections are usually asymptomatic. This is because the parasite has evolved the ability to exist within the brain by dampening the immune response. The parasite can therefore asymptomatically co-exist with its host for years ­ or even an entire lifetime. The strategy has proved so successful that up to one third of the world's population is now thought to be infected with Toxoplasma gondii. While this persistence tends not to be a problem for most healthy individuals, dormant Toxoplasma gondii parasites can reactivate in individuals whose immune systems fail. This can result in life-threatening neurological disease. In pregnant women, Toxoplasma gondii parasites can also cross the placenta, which can trigger miscarriage or cause harmful disease in the newborn. To develop treatments for these cases of symptomatic disease, we need to understand how the parasite hides from the immune system in asymptomatic individuals. Mendez et al. have therefore leveraged a mouse model in which neurons injected with Toxoplasma gondii proteins (Toxoplasma-injected neurons, or 'TINs') produce a green fluorescent protein. This enables the infected cells to be viewed under a microscope. Examining the mouse brains revealed that most TINs were located in two specific regions: the cortex and the striatum. The cortex is the brain's outer layer of tissue. The striatum is a structure deep within the brain that helps regulate movement and responses to rewards. Both the cortex and the striatum contain different types of neurons. The results revealed that the proteins from the parasite were spread roughly equally among the various cell types, rather than targeting a specific subtype of neuron. Neurons close to TINs had slightly abnormal electrical activity, whereas the TINs themselves had highly abnormal activity. By eight weeks post-infection, however, the number of TINS had fallen by around 90%. This suggests that many neurons containing Toxoplasma protein are sick and dying, and that their altered electrical activity reflects this unhealthy state. Understanding how Toxoplasma parasites persist in the brain has the potential to reveal new targets for treating symptomatic infections. It could even provide new possibilities for targeting the inflammation that drives many other neurological diseases. Harnessing this potential will require finding out why Toxoplasma gondii infects specific brain regions and why most neurons that directly interact with the parasite die.

Bridging Computational Vaccinology and Vaccine Development Through Systematic Identification, Characterization, and Downselection of Conserved and Variable Circumsporozoite Protein CD4 T Cell Epitopes From Diverse Plasmodium falciparum Strains.

An effective malaria vaccine must prevent disease in a range of populations living in regions with vastly different transmission rates and protect against genetically-diverse Plasmodium falciparum (Pf) strains. The protective efficacy afforded by the currently licensed malaria vaccine, Mosquirix™, promotes strong humoral responses to Pf circumsporozoite protein (CSP) 3D7 but protection is limited in duration and by strain variation. Helper CD4 T cells are central to development of protective immune responses, playing roles in B cell activation and maturation processes, cytokine production, and stimulation of effector T cells. Therefore, we took advantage of recent in silico modeling advances to predict and analyze human leukocyte antigen (HLA)-restricted class II epitopes from PfCSP - across the entire PfCSP 3D7 sequence as well as in 539 PfCSP sequence variants - with the goal of improving PfCSP-based malaria vaccines. Specifically, we developed a systematic workflow to identify peptide sequences capable of binding HLA-DR in a context relevant to achieving broad human population coverage utilizing cognate T cell help and with limited T regulatory cell activation triggers. Through this workflow, we identified seven predicted class II epitope clusters in the N- and C-terminal regions of PfCSP 3D7 and an additional eight clusters through comparative analysis of 539 PfCSP sequence variants. A subset of these predicted class II epitope clusters was synthesized as peptides and assessed for HLA-DR binding in vitro. Further, we characterized the functional capacity of these peptides to prime and activate human peripheral blood mononuclear cells (PBMCs), by monitoring cytokine response profiles using MIMIC® technology (Modular IMmune In vitro Construct). Utilizing this decision framework, we found sufficient differential cellular activation and cytokine profiles among HLA-DR-matched PBMC donors to downselect class II epitope clusters for inclusion in a vaccine targeting PfCSP. Importantly, the downselected clusters are not highly conserved across PfCSP variants but rather, they overlap a hypervariable region (TH2R) in the C-terminus of the protein. We recommend assessing these class II epitope clusters within the context of a PfCSP vaccine, employing a test system capable of measuring immunogenicity across a broad set of HLA-DR alleles.

Low molecular weight protein tyrosine phosphatase (LMW-PTP2) protein can potentially modulate virulence of the parasite Entamoeba histolytica.

The Entamoeba histolytica parasite is the causative agent of amebiasis, infecting approximately 1% of the world population and causing 100,000 deaths per year. It binds to Fibronectin (FN), activating signaling pathways regulated by kinases and phosphatases. EhLMW-PTPs genes from E. histolytica encode for Low Molecular Weight Tyrosine Phosphatases expressed in trophozoites and amoebic cysts. The role of these phosphatases in the virulence of the parasite has not yet been well characterized. Our results showed a differential expression of the EhLMW-PTPs, at the mRNA and protein levels, in an asynchronous trophozoites culture. Furthermore, we observed that trophozoites transfected that overexpressed EhLMW-PTP2 phagocytized fewer erythrocytes, possibly due to decreased phagocytic cups, and showed deficiencies in adherence to FN and less cytopathic effect. These analyzes suggest that the parasite's EhLMW-PTPs have an essential role in the mechanisms of proliferation, adhesion, and phagocytosis, regulating its pathogenicity.

Antitumour metastasis and the antiangiogenic and antitumour effects of a Eimeria stiedae soluble protein.

Some protozoa (Plasmodium falciparum, Toxoplasma gondii, etc) are used to treat cancer because they can improve tumour-induced immunosuppression. This study aims to evaluate the antitumour effect of Eimeria stiedae oocyst soluble protein (ESSP). ESSP was extracted, and mice were injected with 5 × 105 CT26 cells in the right axilla, and then, 50 µg of ESSP was intraperitoneally injected for 5 continuous days. The effect of ESSP on tumour immunity was detected by flow cytometry 25 days after the CT26 inoculation. The results showed that ESSP can inhibit the growth of CT26 subcutaneous tumours; significantly increase the expression of MHC I, MHC II, CD80 and CD86 on the surface of splenic dendritic cells; and enhance the level of IL-12 secretion. ESSP induced an increase in the number of NK cells in the mouse spleen, and the levels of IFN-γ and CD107 were upregulated in the NK cells and CD8+ T cells. The number of metastatic nodules in the lung tumours in the mice was significantly reduced, and the number of tubes, area of the loops and total length of the tubes were significantly reduced. ESSP enhances the antitumour immune response and inhibits tumour growth, metastasis and angiogenesis.

Toxoplasma GRA16 Inhibits NF-κB Activation through PP2A-B55 Upregulation in Non-Small-Cell Lung Carcinoma Cells.

Nuclear factor kappa B (NF-κB) activation is a well-known mechanism by which chemoresistance to anticancer agents is reported. It is well-known that irinotecan as a chemotherapeutic drug against non-small-cell lung carcinoma (NSCLC) has limited anticancer effect due to NF-κB activation. In this study, we propose the novel role of GRA16, a dense granule protein of Toxoplasma gondii, as an anticancer agent to increase the effectiveness of chemotherapy via the inhibition of NF-κB activation. To demonstrate this, H1299 cells were stably transfected with GRA16. The anticancer effects of GRA16 were demonstrated as a reduction in tumor size in a mouse xenograft model. GRA16 directly elevated B55 regulatory subunit of protein phosphatase 2A (PP2A-B55) expression in tumor cells, thereby decreasing GWL protein levels and ENSA phosphorylation. This cascade, in turn, induced PP2A-B55 activation and suppressed AKT/ERK phosphorylation and cyclin B1 levels, suggesting reduced cell survival and arrested cell cycle. Moreover, PP2A-B55 activation and AKT phosphorylation inhibition led to NF-κB inactivation via the reduction in inhibitory kappa B kinase beta (IKKß) levels, de-phosphorylation of inhibitor of kappa B alpha (IκBα), and reduction in the nuclear transit of NF-κB p65. Furthermore, this molecular mechanism was examined under irinotecan treatment. The PP2A-B55/AKT/NF-κB p65 pathway-mediated anticancer effects were only induced in the presence of GRA16, but not in the presence of irinotecan. Moreover, GRA16 synergistically promoted the anticancer effects of irinotecan via the induction of the sub-G1 phase and reduction of cell proliferation. Collectively, irinotecan and GRA16 co-treatment promotes the anticancer effects of irinotecan via NF-κB inhibition and cell cycle arrest induced by GRA16, subsequently increasing the chemotherapeutic effect of irinotecan to NSCLC cells via NF-κB inhibition.

Immune response and protective efficacy of recombinant Enterococcus faecalis displaying dendritic cell-targeting peptide fused with Eimeria tenella 3-1E protein.

Avian coccidiosis causes significant economic losses on the global poultry breeding industry. Exploration of new-concept vaccines against coccidiosis has gradually become a research hotspot. In this study, an Enterococcus faecalis strain (MDXEF-1) showing excellent performance isolated from chicken intestinal tract was used as a vector to deliver Eimeria target protein. The plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA harboring dendritic cell-targeting peptide (DCpep) fusion with Eimeria tenella NAΔ3-1E gene (3-1E protein-coding gene without start codon ATG and terminator codon TAA) was electrotransformed into MDXEF-1 to generate the recombinant bacteria MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA in which NAΔ3-1E protein was covalently anchored to the surface of bacteria cells by cell wall anchor (CWA) sequence. The expression of target fusion protein DCpep-NAΔ3-1E-CWA was detected by Western blot. Each chicken was immunized 3 times at 2-wk intervals with live E. faecalis expressing DCpep-NAΔ3-1E fusion protein (DCpep-NAΔ3-1E group), live E. faecalis expressing NAΔ3-1E protein (NAΔ3-1E group), and live E. faecalis containing empty vector only. The 3 immunized groups were then challenged with homologous E. tenella sporulated oocyst after immunizations, and the immune response and protective efficacy in each group were evaluated. The results showed that serum IgG levels, secretory IgA levels in cecal lavage, proportion of CD4+ and CD8α+ cells in peripheral blood, and mRNA expression levels of IL-2 and IFN-γ in the spleen were significantly higher in chickens in the DCpep-NAΔ3-1E group than in chickens of the NAΔ3-1E group (P < 0.05). Oral immunization to chickens with live E. faecalis expressing DCpep-NAΔ3-1E offered more protective efficacy against homologous challenge including significant improved body weight gain, increased oocyst decrease ratio, and reduced average lesion scores in cecum compared with chickens with live E. faecalis expressing NAΔ3-1E protein. These results suggest that recombinant E. faecalis expressing dendritic cell-targeting peptide fusion with E. tenella 3-1E protein could be a potential approach for prevention of Eimeria infection.

MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist.

Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.

Production and Characterization of MIC1: A Lectin from Toxoplasma gondii.

Some lectins of pathogens interact with host cells through the recognition of specific carbohydrates displayed on the mammals' cell surface. The microneme protein 1 (MIC1) from Toxoplasma gondii has a lectin domain that specifically binds sialic acid residues, often found in the terminal positions of N-glycans of mammalian cells. The necessary studies on the MIC1 biological roles have been limited initially by the laborious purification of the protein from T. gondii tachyzoites and the low yields verified. Then Escherichia coli has been transformed with a construct containing the MIC1 gene, and the obtained recombinant MIC1 (rMIC1) has been purified from the inclusion bodies. Herein, we detail the methodology of heterologous production and purification of rMIC1 and protocols to assay the rMIC1 lectin ability.

A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine.

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.

GK1 Improves the Immune Response Induced by Dendritic Cells of BALB/c Mice Infected with Leishmania mexicana Promastigotes.

PURPOSE: Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), and their capacity to activate the immune response has been widely used in immunotherapies against different diseases, predominantly cancer. However, they have not been so widely used in immunotherapies against infectious diseases. Leishmania mexicana is the causative agent of cutaneous leishmaniasis in Mexico, which can result in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). DCL is characterized by the incapability of the immune response to control the parasite, which thus disseminates to all teguments. Treatments against DCL have shown low efficacy, which is a reason why alternative therapies such as immunotherapies are promising. One adjuvant that has proven its effectiveness in immunotherapies against some cancers and infections is GK1, a component of the SPVac vaccine against porcine cysticercosis. GK1 has the capacity to elicit proinflammatory cytokines and chemokines from DCs and macrophages. METHODS: We pulsed bone marrow-derived dendritic cells (BMDCs) with GK1 and a lysate obtained from L. mexicana promastigotes and tested the efficacy of this combination against the infection of susceptible mice with L. mexicana. RESULTS: We found that BMDCs stimulated with GK1 and a lysate of L. mexicana promastigotes secreted IFN-γ and IL-12, and when they were adoptively transferred to BALB/c mice which were then infected with L. mexicana promastigotes, there was a reduction in the size of the lesion and in the parasite load. CONCLUSIONS: The adjuvant properties of GK1 along with parasite antigens may have a protective effect against the infection of BALB/c mice with L. mexicana.

The Plasmodium falciparum circumsporozoite protein produced in Lactococcus lactis is pure and stable.

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis-derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.