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Graphical abstract, Highlights • Current vaccine candidates against helminth infection have shown limited success. • Helminths release extracellular vesicles (EVs) which act on host cells and are a rich source of antigens for new vaccines. • The biogenesis, release and immunomodulatory functions of helminth EVs are reviewed. • Utilisation of EVs in vaccine generation are discussed, including potential antigens and routes of delivery., The hunt for effective vaccines against the major helminth diseases of humans has yet to bear fruit despite much effort over several decades. No individual parasite antigen has proved to elicit full protective immunity, suggesting that combinatorial strategies may be required. Recently it has been discovered that extracellular vesicles released by parasitic helminths contain multiple potential immune modulators, which could together be targeted by a future vaccine. Increasing knowledge of helminth extracellular vesicle components, both enclosed by and exposed on the membrane, will open up a new field of targets for an effective vaccine. This review discusses the interactions between helminth extracellular vesicles and the immune system discovered thus far, and the advantages of targeting these lipid-bound packages with a vaccine. In addition, we also comment upon specific antigens that may be the best targets for an anti-helminth vaccine. In the future, extensive knowledge of the parasites' full arsenal in controlling their host may finally provide us with the ideal target for a fully effective vaccine.

Racemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment., Author summary Racemose neurocysticercosis is the most aggressive and lethal form of the disease characterized by progressive expansion of the larval stage of Taenia solium within the subarachnoid spaces of the brain. Currently, the cellular or molecular events that promote its formation and development are unknown. Our observations, employing tissue samples, revealed the presence of mitotically active cells in the bladder wall of the racemose larvae. Finally, we isolated and established primary cell cultures and observed that these cells are sensitive (proliferate in response to insulin). These results suggest that the proliferative cells stimulated by host biomolecules like hormones would be responsible for the abnormal growth of racemose larvae. Further studies are needed to identify all genes and pathways altered in the racemose form. The primary cell cultures will be used as a therapeutic target and allow us in future works to understand the developmental biology of the parasite.

[Background]: Freezing is considered the most suitable technological treatment to avoid Anisakis infection from eating raw or undercooked fish but modifications of their cuticles upon freezing may reduce their resistance to gastric fluids, provoking a greater release of allergens. This work aimed to study the relationship between freezing-induced modifications of Anisakis simplex s.l., antigen recognition, and resistance to oral and gastric digestion in spiked fish mince., [Results]: (i) Differences between non-treated larvae and larvae that survived freezing / thawing were studied in terms of respiratory capacity, survival in simulated gastric fluid (SGF), recognition of antigens and allergens. (ii) Untreated (i.e. chilled) mince containing live larvae, mince frozen at two freezing rates, with a negative (uninfected) mince and a positive mince (infected with broken larvae) as controls, were subjected to the oral and gastric phases of a simulated digestion process. Anisakis able to survive freezing showed lower resistance to gastric fluid (i.e. faster mortality as compared to controls). Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in SGF. In treatments rendering complete larvae mortality, the highest loss of larvae integrity was found upon fast freezing. There was a positive correlation between antigen release and the number of ruptures of larvae after the oral digestion phase, whereas a more complex trend was observed after oral plus gastric digestion phases., [Conclusion]: These results suggest a new factor to consider for sensitized patients and suggest that the numbers of L3 should be reduced before industrial freezing to minimize risk., This work has been financed by the Spanish ANIRISK (AGL2015‐68248‐C2‐1‐R) MINECO/FEDER.

Cryptosporidium spp. is a zoonotic intracellular protozoan and a significant cause of diarrhoea in humans and animals worldwide. This parasite can cause high morbidity in immunocompromised people and children in developing countries, livestock being the main reservoir. This study was aimed at performing preliminary tests on Swiss albino weaned mice (ICR) to evaluate the humoral immune response induced against peptides derived from Cryptosporidium parvum CP15 (15 kDa sporozoite surface antigen) and CSL (circumsporozoite-like antigen) proteins. Peptides were identified and characterised using bioinformatics tools and were chemically synthesised. The antibody response was determined and the neutralising effect of antibodies was measured in cell culture. Despite all peptides studied here were capable of stimulating antibody production, neutralising antibodies were detected for just two of the CP15-derived ones. Additional studies aimed at evaluating further the potential of such peptides as vaccine candidates are thus recommended. © 2018 Elsevier Ltd

Like Thomas Hardy's famous novel Far from the Madding Crowd, Plasmodium falciparum parasites display their most relevant survival structures (proteins) involved in host cell invasion far away from the immune system's susceptible regions, displaying tremendous genetic variability, to attract the immune response and escape immune pressure. The 3D structure localisation of the conserved amino acid sequences of this deadly parasite's most relevant proteins involved in host cell invasion, as well as the location of the highly polymorphic, highly immunogenic regions, clearly demonstrates that such structures are far apart, sometimes 90&#176, to 180&#176, opposite, thereby rendering the immune response useless. It is also shown here that these conserved, functionally-relevant structures are immunologically silent, since no immune response has been induce

Porcine cysticercosis is an endemic parasitic disease caused by infection with Taenia solium that is found predominantly in developing countries. In order to aid in the development of simple diagnostic approaches, identification and characterization of potential new antigens for immunodiagnostic purposes is desired. The cysteine protease family has previously been found to have important immunodiagnostic properties. These proteases are expressed as zymogens which contain a signal peptide, pro-peptide, and an active domain. Subsequent catalytic cleavage of the pro-peptide converts these zymogens into enzymes. With the use of bioinformatic tools we identified an active domain of a novel cathepsin L-like cysteine protease (TsolCL) in the T. solium genome. The TsolCL gene includes 705 nucleotides (nt) within a single intron and a 633 nt exonic sequence encoding an active protein of 211 amino acids. Sequence alignment and phylogenetic analysis suggest that the TsolCL gene is closely related to genes found in Echinoccocus granulosus and E. multiloculars. In addition, TsolCL was found to have a 61.9%–99.0% similarity to other cathepsin L proteins found in other helminths and mammals. We cloned, expressed, purified, and characterized the recombinant active TsolCL (27 kDa) using the baculovirus-insect cell expression system. TsolCL showed cysteine protease enzymatic activity with the capacity to hydrolyze the Z-Phe-Arg-AMC substrate as well as bovine serum albumin. However, TsolCL was not able to hydrolyze human immunoglobulin. In addition, TsolCL has cathepsin L conserved amino acid residues in the catalytic site (Gln8, Cys14, His159, Asn179 and Trp181) and the motif GCNGG. Using ELISA, TsolCL was able to distinguish circulating IgG antibodies between healthy animals and naturally infected pigs with cysticercosis, showing a moderate sensitivity of 83.33% (40/48; 95% CI: [69.8%–92.5 %]), and a specificity of 83.78% (31/37; 95% CI: [67.9%–93.8%]). In conclusion, a novel cathepsin L-like cysteine protease from a T. solium metacestode was expressed successfully in Baculovirus system and was evaluated as a candidate antigen to diagnose porcine cysticercosis using the ELISA immunoassay.

Malaria caused by Plasmodium vivax is a neglected disease which is responsible for the highest morbidity in both Americas and Asia. Despite continuous public health efforts to prevent malarial infection, an effective antimalarial vaccine is still urgently needed. P. vivax vaccine development involves analyzing naturally-infected patients' immune response to the specific proteins involved in red blood cell invasion. The P. vivax rhoptry neck protein 2 (PvRON2) is a highly conserved protein which is expressed in late schizont rhoptries; it interacts directly with AMA-1 and might be involved in moving-junction formation. Bioinformatics approaches were used here to select B- and T-cell epitopes. Eleven high-affinity binding peptides were selected using the NetMHCIIpan-3.0 in silico prediction tool; their in vitro binding to HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101 or HLA-DRB1*1302 was experimentally assessed. Four peptides (39152 (HLA-DRB1*04 and 11), 39047 (HLA-DRB1*07), 39154 (HLADRB1*13) and universal peptide 39153) evoked a naturally-acquired T-cell immune response in P. vivax-exposed individuals from two endemic areas in Colombia. All four peptides had an SI greater than 2 in proliferation assays; however, only peptides 39154 and 39153 had significant differences compared to the control group. Peptide 39047 was able to significantly stimulate TNF and IL-10 production while 39154 stimulated TNF production. Allele-specific peptides (but not the universal one) were able to stimulate IL-6 production; however, none induced IFN-? production. The Bepipred 1.0 tool was used for selecting four B-cell epitopes in silico regarding humoral response. Peptide 39041 was the only one recognized by P. vivax-exposed individuals' sera and had significant differences concerning IgG subclasses; an IgG2 > IgG4 profile was observed for this peptide, agreeing with a protection-inducing role against P. falciparum and P. vivax as previously described for antigens such as RESA and MSP2. The bioinformatics results and in vitro evaluation reported here highlighted two T-cell epitopes (39047 and 39154) being recognized by memory cells and a B-cell epitope (39041) identified by P. vivax-exposed individuals' sera which could be used as potential candidates when designing a subunit-based vaccine. © 2018 López, Yepes-Pérez, Díaz-Arévalo, Patarroyo and Patarroyo.

Malaria is an infectious disease caused by parasites from the genus Plasmodium (P. falciparum and P. vivax are responsible for 90% of all clinical cases); it is widely distributed throughout the world’s tropical and subtropical regions. The P. vivax Pv12 protein is involved in invasion, is expressed on merozoite surface and has been recognised by antibodies from individuals exposed to the disease. In this study, B- and T-cell epitopes from Pv12 were predicted and characterised to advance in the design of a peptide-based vaccine against malaria. For evaluating the humoral response of individuals exposed to natural P. vivax infection from two endemic areas in Colombia, BepiPred-1.0 software was used for selecting B-cell epitopes. B-cell epitope 39038 displayed the greatest recognition by naturally-acquired antibodies and induced an IgG2/IgG4 response. NetMHCIIpan-3.1 prediction software was used for selecting peptides having high affinity binding for HLA-DRβ1* allele lineages and this was confirmed by in-vitro binding assays. T-epitopes 39113 and 39117 triggered a memory T-cell response (Stimulation Index2) and significant cytokine production. Combining in-silico, in-vitro and functional assays, two Pv12 protein regions (containing peptides 39038, 39040, 39113 and 39117) have thus been characterised as promising vaccine candidates against P. vivax malaria. © 2018 Yepes-Perez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Rational strategies for obtaining malaria vaccine candidates should include not only a proper selection of target antigens for antibody stimulation, but also a versatile molecular design based on ordering the right pieces from the complex pathogen molecular puzzle towards more active and functional immunogens. Classical Plasmodium falciparum antigens regarded as vaccine candidates have been selected as model targets in this study. Among all possibilities we have chosen epitopes of PfCSP, STARP; MSA1 and Pf155/RESA from pre- A nd erythrocyte stages respectively for designing a large 82-residue chimeric immunogen. A number of options aimed at diminishing steric hindrance for synthetic procedures were assessed based on standard Fmoc chemistry such as building block orthogonal ligation; pseudo-proline and microwave-assisted procedures, therefore the large-chimeric target was produced, characterized and immunologically tested. Antigenicity and functional in vivo efficacy tests of the large-chimera formulations administered alone or as antigen mixtures have proven the stimulation of high antibody titers, showing strong correlation with protection and parasite clearance of vaccinated BALB/c mice after being lethally challenged with both P. berghei-ANKA and P. yoelii 17XL malaria strains. Besides, 3D structure features shown by the large-chimera encouraged as to propose using these rational designed large synthetic molecules as reliable vaccine candidate-presenting systems.

Background Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. Results Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. Conclusion The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended. Electronic supplementary material The online version of this article (doi: 10.1186/s13071-017-2549-y) contains supplementary material, which is available to authorized users.

Background Plasmodium vivax uses multiple ligand-receptor interactions for preferential invasion of human reticulocytes. Several of these ligands have been identified by in silico approaches based on the role displayed by their orthologs in other Plasmodium species during initial adhesion or invasion. However, the cell adhesion role of proteins that are exclusive to species that specifically invade reticulocytes (as P. vivax and P. cynomolgi) has not been evaluated to date. This study aimed to characterise an antigen shared between Plasmodium species that preferentially infect reticulocytes with a focus on assessing its binding activity to target cells. Results An in silico analysis was performed using P. vivax proteome data to identify and characterise one antigen shared between P. vivax and P. cynomolgi. This led to identification of the pvrbsa gene present in the P. vivax VCG-I strain genome. This gene is transcribed in mature schizonts and encodes a protein located on the parasite surface. rPvRBSA was antigenic and capable of binding to a population of reticulocytes with a different Duffy phenotype. Interestingly, the molecule showed a higher percentage of binding to immature human reticulocytes (CD71hi). Conclusions This study describes for the first time, a molecule involved in host cell binding that is exclusive in reticulocyte-infecting Plasmodium species. This suggest that PvRBSA is an antigenic adhesin that plays a role in parasite binding to target cells. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2185-6) contains supplementary material, which is available to authorized users.

Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes. © 2017 Macmillan Publishers Limited. All rights reserved.

A totally effective, antimalarial vaccine must involve sporozoite and merozoite proteins (or their fragments) to ensure complete parasite blocking during critical invasion stages. This Special Report examines proteins involved in critical biological functions for parasite survival and highlights the conserved amino acid sequences of the most important proteins involved in sporozoite invasion of hepatocytes and merozoite invasion of red blood cells. Conserved high activity binding peptides are located in such proteins' functionally strategic sites, whose functions are related to receptor binding, nutrient and protein transport, enzyme activity and molecule-molecule interactions. They are thus excellent targets for vaccine development as they block proteins binding function involved in invasion and also their biological function. © 2017 Future Medicine Ltd.

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte-binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba-140, eba-175, eba-181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba-140 seemed to be under stronger diversifying selection in South America than eba-175. In contrast, eba-181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins. © 2014 John Wiley & Sons Ltd.

Chagas disease is a complex zoonotic pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite presents remarkable genetic variability and has been grouped into six discrete typing units (DTUs). The association between the DTUs and clinical outcome remains unknown. Chagas disease and co-infection with HIV/AIDS has been reported widely in Brazil and Argentina. Herein, we present the molecular analyses from a Chagas disease patient with HIV/AIDS co-infection in Colombia who presented severe cardiomyopathy, pleural effusion, and central nervous system involvement. A mixed infection by T. cruzi genotypes was detected. We suggest including T. cruzi in the list of opportunistic pathogens for the management of HIV patients in Colombia. The epidemiological implications of this finding are discussed. © 2014 The Authors.

To block the different stages of Plasmodium falciparum invasion into human hepatocytes and red blood cells, we have focused on those proteins belonging to the pre-erythrocytic stage. One of these proteins is Sporozoite Threonine and Asparagine Rich Protein (STARP), which is a ligand used by P. falciparum parasites to bind Hepatic cells (HepG2). Previous studies on this protein identified two conserved peptides binding with high activity to HepG2 cells (namely 20546 and 20570) with corresponding critical hepatic-cell binding residues and determined an important role for these two peptides in the invasion process. This study shows the results of immunization trials in Aotus monkeys with native STARP peptides and analogues modified in critical hepatic-cell binding residues. The results show that native peptides are not immunogenic but can induce high-antibody titers when their critical residues are replaced by other with similar volume and mass but different polarity. Nuclear Magnetic Resonance (1H NMR) studies revealed that native peptides (non-immunogenic) displayed shorter ?-helical regions compared to their highly immunogenic modified analogues. Binding assays with HLA-DR?1* molecules showed that 20546 modified peptides inducing high-antibody titers (24972, 24320 and 24486) bound to HLA-DR?1*0301 molecules, while the 20570 modified analogue (24322) bound to HLA-DR?1*0101. The results support including these high-immunogenic STARP-derived modified peptides as pre-erythrocytic candidates to be included in the design of a synthetic antimalarial vaccine. © 2010 Elsevier Ltd.

Based on the 3D X-ray crystallographic structures of relevant proteins of the malaria parasite involved in invasion to host cells and 3D NMR structures of High Activity Binding Peptides (HABPs) and their respective analogues, it was found that HABPs are rendered into highly immunogenic and sterile immunity inducers in the Aotus experimental model by modifying those amino acids that establish H-bonds with other HABPs or binding to host's cells. This finding adds striking and novel physicochemical principles, at the atomic level, for a logical and rational vaccine development methodology against infectious disease, among them malaria. © 2010 Elsevier Inc. All rights reserved.