The mRNA Vaccine Technology Era and the Future Control of Parasitic Infections.

Despite intensive long-term efforts, with very few exceptions, the development of effective vaccines against parasitic infections has presented considerable challenges, given the complexity of parasite life cycles, the interplay between parasites and their hosts, and their capacity to escape the host immune system and to regulate host immune responses. For many parasitic diseases, conventional vaccine platforms have generally proven ill suited, considering the complex manufacturing processes involved and the costs they incur, the inability to posttranslationally modify cloned target antigens, and the absence of long-lasting protective immunity induced by these antigens. An effective antiparasite vaccine platform is required to assess the effectiveness of novel vaccine candidates at high throughput. By exploiting the approach that has recently been used successfully to produce highly protective COVID mRNA vaccines, we anticipate a new wave of research to advance the use of mRNA vaccines to prevent parasitic infections in the near future. This article considers the characteristics that are required to develop a potent antiparasite vaccine and provides a conceptual foundation to promote the development of parasite mRNA-based vaccines. We review the recent advances and challenges encountered in developing antiparasite vaccines and evaluate the potential of developing mRNA vaccines against parasites, including those causing diseases such as malaria and schistosomiasis, against which vaccines are currently suboptimal or not yet available.

Transmission-Blocking Vaccines against Schistosomiasis Japonica.

Control of schistosomiasis japonica, endemic in Asia, including the Philippines, China, and Indonesia, is extremely challenging. Schistosoma japonicum is a highly pathogenic helminth parasite, with disease arising predominantly from an immune reaction to entrapped parasite eggs in tissues. Females of this species can generate 1000-2200 eggs per day, which is about 3- to 15-fold greater than the egg output of other schistosome species. Bovines (water buffalo and cattle) are the predominant definitive hosts and are estimated to generate up to 90% of parasite eggs released into the environment in rural endemic areas where these hosts and humans are present. Here, we highlight the necessity of developing veterinary transmission-blocking vaccines for bovines to better control the disease and review potential vaccine candidates. We also point out that the approach to producing efficacious transmission-blocking animal-based vaccines before moving on to human vaccines is crucial. This will result in effective and feasible public health outcomes in agreement with the One Health concept to achieve optimum health for people, animals, and the environment. Indeed, incorporating a veterinary-based transmission vaccine, coupled with interventions such as human mass drug administration, improved sanitation and hygiene, health education, and snail control, would be invaluable to eliminating zoonotic schistosomiasis.

Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase.

Background: Recent studies on CRISPR/Cas9-mediated gene editing in Schistosoma mansoni have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest. Methods: To improve the efficiency of CRISPR/Cas9 genome editing in schistosomes, we used lentivirus, which has been effectively used for gene editing in mammalian cells, to deliver plasmid DNA encoding Cas9 nuclease, a sgRNA targeting acetylcholinesterase (SmAChE) and a mCherry fluorescence marker into schistosomes. Results: MCherry fluorescence was observed in transduced eggs, schistosomula, and adult worms, indicating that the CRISPR components had been delivered into these parasite stages by lentivirus. In addition, clearly changed phenotypes were observed in SmAChE-edited parasites, including decreased SmAChE activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transduction-based CRISPR/Cas9 gene modifications in SmAChE-edited schistosomes were homology-directed repair predominant but with much lower efficiency than that obtained using electroporation (data previously published by our laboratory) for the delivery of CRISPR components. Conclusion: Taken together, electroporation is more efficient than lentiviral transduction in the delivery of CRISPR/Cas9 into schistosomes for programmed genome editing. The exploration of tactics for enhancing CRISPR/Cas9 gene editing provides the basis for the future improvement of programmed genome editing in S. mansoni.

CRISPR/Cas9: A new tool for the study and control of helminth parasites.

Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9-targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9-mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.

CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.

Whole-genome sequence of the bovine blood fluke Schistosoma bovis supports interspecific hybridization with S. haematobium.

Mesenteric infection by the parasitic blood fluke Schistosoma bovis is a common veterinary problem in Africa and the Middle East and occasionally in the Mediterranean Region. The species also has the ability to form interspecific hybrids with the human parasite S. haematobium with natural hybridisation observed in West Africa, presenting possible zoonotic transmission. Additionally, this exchange of alleles between species may dramatically influence disease dynamics and parasite evolution. We have generated a 374 Mb assembly of the S. bovis genome using Illumina and PacBio-based technologies. Despite infecting different hosts and organs, the genome sequences of S. bovis and S. haematobium appeared strikingly similar with 97% sequence identity. The two species share 98% of protein-coding genes, with an average sequence identity of 97.3% at the amino acid level. Genome comparison identified large continuous parts of the genome (up to several 100 kb) showing almost 100% sequence identity between S. bovis and S. haematobium. It is unlikely that this is a result of genome conservation and provides further evidence of natural interspecific hybridization between S. bovis and S. haematobium. Our results suggest that foreign DNA obtained by interspecific hybridization was maintained in the population through multiple meiosis cycles and that hybrids were sexually reproductive, producing viable offspring. The S. bovis genome assembly forms a highly valuable resource for studying schistosome evolution and exploring genetic regions that are associated with species-specific phenotypic traits.

The mitochondrial genome of Angiostrongylus mackerrasae is distinct from A. cantonensis and A. malaysiensis.

The native rat lungworm (Angiostrongylus mackerrasae) and the invasive rat lungworm (Angiostrongylus cantonensis) occur in eastern Australia. The species identity of A. mackerrasae remained unquestioned until relatively recently, when compilation of mtDNA data indicated that A. mackerrasae sensu Aghazadeh et al. (2015b) clusters within A. cantonensis based on their mitochondrial genomes (mtDNA). To re-evaluate the species identity of A. mackerrasae, we sought material that would be morphologically conspecific with A. mackerrasae. We combined morphological and molecular approaches to confirm or refute the specific status of A. mackerrasae. Nematodes conspecific with A. mackerrasae from Rattus fuscipes and Rattus rattus were collected in Queensland, Australia. Morphologically identified A. mackerrasae voucher specimens were characterized using amplification of cox1 followed by the generation of reference complete mtDNA. The morphologically distinct A. cantonensis, A. mackerrasae and A. malaysiensis are genetically distinguishable forming a monophyletic mtDNA lineage. We conclude that A. mackerrasae sensu Aghazadeh et al. (2015b) is a misidentified specimen of A. cantonensis. The availability of the mtDNA genome of A. mackerrasae enables its unequivocal genetic identification and differentiation from other Angiostrongylus species.

Use of kinase inhibitors against schistosomes to improve and broaden praziquantel efficacy.

Praziquantel (PZQ) is the drug of choice for schistosomiasis. The potential drug resistance necessitates the search for adjunct or alternative therapies to PZQ. Previous functional genomics has shown that RNAi inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) gene in Schistosoma adult worms significantly improved the effectiveness of PZQ. Here we tested the in vitro efficacy of 15 selective and non-selective CaMK inhibitors against Schistosoma mansoni and showed that PZQ efficacy was improved against refractory juvenile parasites when combined with these CaMK inhibitors. By measuring CaMK activity and the mobility of adult S. mansoni, we identified two non-selective CaMK inhibitors, Staurosporine (STSP) and 1Naphthyl PP1 (1NAPP1), as promising candidates for further study. The impact of STSP and 1NAPP1 was investigated in mice infected with S. mansoni in the presence or absence of a sub-lethal dose of PZQ against 2- and 7-day-old schistosomula and adults. Treatment with STSP/PZQ induced a significant (47-68%) liver egg burden reduction compared with mice treated with PZQ alone. The findings indicate that the combination of STSP and PZQ dosages significantly improved anti-schistosomal activity compared to PZQ alone, demonstrating the potential of selective and non-selective CaMK/kinase inhibitors as a combination therapy with PZQ in treating schistosomiasis.

Glycans in the roles of parasitological diagnosis and host-parasite interplay.

The investigation of the glycan repertoire of several organisms has revealed a wide variation in terms of structures and abundance of glycan moieties. Among the parasites, it is possible to observe different sets of glycoconjugates across taxa and developmental stages within a species. The presence of distinct glycoconjugates throughout the life cycle of a parasite could relate to the ability of that organism to adapt and survive in different hosts and environments. Carbohydrates on the surface, and in excretory-secretory products of parasites, play essential roles in host-parasite interactions. Carbohydrate portions of complex molecules of parasites stimulate and modulate host immune responses, mainly through interactions with specific receptors on the surface of dendritic cells, leading to the generation of a pattern of response that may benefit parasite survival. Available data reviewed here also show the frequent aspect of parasite immunomodulation of mammalian responses through specific glycan interactions, which ultimately makes these molecules promising in the fields of diagnostics and vaccinology.

Gene Expression in Developmental Stages of Schistosoma japonicum Provides Further Insight into the Importance of the Schistosome Insulin-Like Peptide.

We showed previously that the Schistosoma japonicum insulin-like peptide (SjILP) binds the worm insulin receptors, thereby, activating the parasite's insulin pathway and emphasizing its important role in regulating uptake of glucose, a nutrient essential for parasite survival. Here we show that SjILP is differentially expressed in the schistosome life cycle and is especially highly transcribed in eggs, miracidia, and adult female worms. RNA inference was employed to knockdown SjILP in adults in vitro, with suppression confirmed by significantly reduced protein production, declined adenosine diphosphate levels, and reduction in glucose consumption. Immunolocalization showed that SjILP is located to lateral gland cells of mature intra-ovular miracidia in the schistosome egg, and is distributed on the ciliated epithelium and internal cell masses of newly transformed miracidia. In schistosomula, SjILP is present on the tegument in two antero-lateral points, indicating highly polarized expression during cercarial transformation. Analysis of serum from S. japonicum-infected mice by ELISA using a recombinant form of SjILP as an antigen revealed IgG immunoreactivity to this molecule at 7 weeks post-infection indicating it is likely secreted from mature eggs into the host circulation. These findings provide further insights on ILP function in schistosomes and its essential roles in parasite survival and growth in different development stages.

Calcium and Ca2+/Calmodulin-dependent kinase II as targets for helminth parasite control.

In eukaryotes, effective calcium homeostasis is critical for many key biological processes. There is an added level of complexity in parasites, particularly multicellular helminth worms, which modulate calcium levels while inhabiting the host microenvironment. Parasites ensure efficient calcium homeostasis through gene products, such as the calmodulin-dependent kinases (CaMK), the main focus of this review. The importance of CaMK is becoming increasingly apparent from recent functional studies of helminth and protozoan parasites. Investigations on the molecular regulation of calcium and the role of CaMK are important for both supplementing current drug regimens and finding new antiparasitic compounds. Whereas calcium regulators, including CaMK, are well characterised in mammalian systems, knowledge of their functional properties in parasites is increasing but is still in its infancy.

Optimization of the Helmintex method for schistosomiasis diagnosis.

A diagnostic test that is reliable, sensitive, and applicable in the field is extremely important in epidemiological surveys, during medical treatment for schistosomiasis, and for the control and elimination of schistosomiasis. The Helmintex (HTX) method is based on the use of magnetic beads to trap eggs in a magnetic field. This technique is highly sensitive, but the screening of fecal samples consumes lots of time, thus delaying the results, especially in field studies. The objective of this work was to determine the effects of incorporation of the detergent Tween-20 into the method in an attempt to decrease the final pellet volume produced by the HTX method as well as the use of ninhydrin to stain the Schistosoma mansoni eggs. We showed that these modifications reduced the final volume of the fecal sediment produced in the last step of the HTX method by up to 69% and decreased the screening time to an average of 10.1 min per sample. The use of Tween 20 and ninhydrin led to a high percentage of egg recovery (27.2%). The data obtained herein demonstrate that the addition of detergent and the use of ninhydrin to the HTX process can optimize the screening step and also improve egg recovery, thus justifying the insertion of these steps into the HTX method.

Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.

This study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.

A Plasmodium falciparum S33 proline aminopeptidase is associated with changes in erythrocyte deformability.

Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PfPAP). Unlike other P. falciparum aminopeptidases, PfPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PfPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PfPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies.

Fine structure of uterus and non-functioning paruterine organ in Orthoskrjabinia junlanae (Cestoda, Cyclophyllidea).

Some cyclophyllidean cestodes provide protection for their eggs in the external environment by providing them with additional protective layers around the egg membranes. In attempting to examine such adaptations, the microanatomy and fine structure of the uterus of pregravid and gravid proglottids of the cyclophyllidean cestode Orthoskrjabinia junlanae, a parasite of mammals that inhabit a terrestrial but moist environment, were studied. In the initial stages of uterine development, developing embryos locate freely in the lumen of a saccate uterus that later partitions into chambers. Each chamber that forms encloses several embryos. The chambers are surrounded by muscle cells that synthesize extracellular matrix actively. The paruterine organs consist of stacks of flattened long outgrowths of muscular cells, interspersed with small lipid droplets. In the gravid proglottids, the size of paruterine organ increases and consists of flattened basal and small rounded apical parts separated by constrictions. The fine structure of the organ wall remains the same: sparse nuclei and stacks of flattened cytoplasmic outgrowths but internal invaginations or lumen in the paruterine organ are absent. Completely developed eggs remain localized in the uterus. Based on the comparative morpho-functional analysis of uterine and paruterine organs and uterine capsules in cestodes, we conclude that these non-functioning paruterine organ in O. junlanae is an example of an atavism. We postulate that the life cycle of the parasite, which infects mammals living in wet habitats, where threats of desiccation of parasite ova is reduced, has favoured a reversion to a more ancestral form of uterine development.

Exploring the molecular mechanisms of parasite-host interactions with a view towards new therapeutics and vaccines.

Despite the massive disease burden worldwide caused by parasitic nematodes and other infectious pathogens, the molecular basis of many infectious diseases caused by these pathogens has been unduly neglected for a long time. Therefore, accelerated progress towards novel therapeutics, and ultimately control of such infectious diseases, is of crucial importance. Capitalising on the wealth of data becoming available from proteomic and genomic studies, new protein targets at the pathogen-host interface can be identified and subjected to protein-based explorations of the molecular basis of pathogen-host interactions. By combining the use of systems and structural biology methodologies, insights into the structural and molecular mechanisms of these interactions can assist in the development of therapeutics and/or vaccines. This brief review examines two different proteins from the body wall of blood flukes - annexins and the stress-induced phosphoprotein 1 - both of which are presently interesting targets for the development of therapeutics.

Carcinogenic Liver Fluke Secretes Extracellular Vesicles That Promote Cholangiocytes to Adopt a Tumorigenic Phenotype.

BACKGROUND: Throughout Asia, there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes, including chronic inflammation and secretion of parasite proteins into the biliary epithelium, drive infection toward cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown. METHODS: Various microscopy techniques were used to identify O. viverrini extracellular vesicles (EVs) and their internalization by human cholangiocytes. Using mass spectrometry we characterized the EV proteome and associated changes in cholangiocytes after EV uptake, and we detected EV proteins in bile of infected hamsters and humans. Cholangiocyte proliferation and interleukin 6 (IL-6) secretion was measured to assess the impact of EV internalization. RESULTS: EVs were identified in fluke culture medium and bile specimens from infected hosts. EVs internalized by cholangiocytes drove cell proliferation and IL-6 secretion and induced changes in protein expression associated with endocytosis, wound repair, and cancer. Antibodies to an O. viverrini tetraspanin blocked EV uptake and IL-6 secretion by cholangiocytes. CONCLUSIONS: This is the first time that EVs from a multicellular pathogen have been identified in host tissues. Our findings imply a role for O. viverrini EVs in pathogenesis and highlight an approach to vaccine development for this infectious cancer.

Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.

Defining a pro-inflammatory neutrophil phenotype in response to schistosome eggs.

Neutrophils contribute to the pathological processes of a number of inflammatory disorders, including rheumatoid arthritis, sepsis and cystic fibrosis. Neutrophils also play prominent roles in schistosomiasis japonica liver fibrosis, being central mediators of inflammation following granuloma formation. In this study, we investigated the interaction between Schistosoma japonicum eggs and neutrophils, and the effect of eggs on the inflammatory phenotype of neutrophils. Our results showed significant upregulated expression of pro-inflammatory cytokines (IL-1α, IL-1ß and IL-8) and chemokines (CCL3, CCL4 and CXCL2) in neutrophils after 4 h in vitro stimulation with S. japonicum eggs. Furthermore, mitochondrial DNA was released by stimulated neutrophils, and induced the production of matrix metalloproteinase 9 (MMP-9), a protease involved in inflammation and associated tissue destruction. We also found that intact live eggs and isolated soluble egg antigen (SEA) triggered the release of neutrophil extracellular traps (NETs), but, unlike those reported in bacterial or fungal infection, NETs did not kill schistosome eggs in vitro. Together these show that S. japonicum eggs can induce the inflammatory phenotype of neutrophils, and further our understanding of the host-parasite interplay that takes place within the in vivo microenvironment of schistosome-induced granuloma. These findings represent novel findings in a metazoan parasite, and confirm characteristics of NETs that have until now, only been observed in response to protozoan pathogens.

Fine structure of the copulatory apparatus of the tapeworm Tetrabothrius erostris (Cestoda: Tetrabothriidea).

The organization and fine structure of the complex copulatory apparatus of Tetrabothrius erostris (Tetrabothriidea) is investigated by light and transmission electron microscopy. A diversity of microstructures was found on the surface of genital ducts. The apical surfaces of male gonadoducts possess tubular and blade-like microtriches that have specific structure in each section of the duct. The apical part of the tubular microtriches contains numerous constrictions in the proximal section of the sperm duct; blade-like microtriches of cirrus possess longitudinal striation in the apical part, and their basal part is reinforced with electron-dense strands. Two types of microtriches occur on the surface of cirrus, and their presence may be considered as systematic features. Prostate glands containing granules of medium electron density (up to 130 nm diameter) are localized in the cirrus sac. The genital atrium contains numerous non-ciliated receptors. Paramyosin-like fibers (up to 200 nm) were found in the muscle fibers surrounding the male atrium canal. Microtriches on the surface of the distal region of the male atrial canal are covered by a glycocalyx. Electron-dense, membrane-like structures (up to 40 nm) lie under the apical membrane of the genital atrium and vagina. These structures do not form a continuous layer; its edges turn down and sink into the apical invaginations of epithelium. Hypotheses on the possible ways of copulation in T. erostris based on the observed ultrastructure are discussed.