Extracellular vesicles isolated from Toxoplasma gondii induce host immune response

This study established a protocol to purify Toxoplasma gondii tachyzoite microvesicles and exosomes, called as extracellular vesicles (EVs). In addition, the investigations were conducted to determine the kinetic of EV release by tachyzoites and whether EV proteins are able to modulate the host immune response. The particle size and concentration released by tachyzoites in culture medium at different incubation‐period were characterized by nanoparticle tracking analysis. Tachyzoites (1 × 10⁶) released around 4.37 ± 0.81 × 10⁸ EVs/mL/h, with size varying between 138.2 and 171.9 nm. EVs released into the medium were purified by gel‐exclusion chromatography and screened by ELISA, using a pool of human positive sera for toxoplasmosis. EV‐fractions contained high concentration of proteins, and EVs were analyzed by scanning and transmission electron microscopies. Tachyzoites released EVs into the culture medium throughout all membrane surface, and these vesicles contain small RNAs/miRNA. Pooled sera from chronically infected human or mice (infected with 2 different T. gondii strains) recognized distinct EV electrophoretic patterns in immunoblotting. T. gondii EVs significantly induced IL‐10, TNF‐α and iNOS in murine macrophages. In conclusion, this study shows that T. gondii secrete/excrete EVs (microvesicles and exosomes) contain miRNA and they were immunologically recognized by host immune response.     

Increased susceptibility to oral Trichuris muris infection in the specific absence of CXCR5+ CD11c+ cells

Trichuris muris is a natural mouse helminth pathogen which establishes infection specifically in the caecum and proximal colon. The rapid expulsion of T. muris in resistant mouse strains is associated with the induction of a protective T helper cell type 2 (Th2)‐polarized immune response. Susceptible mouse strains, in contrast, mount an inappropriate Th1 response to T. muris infection. Expression of the chemokine CXCL13 by stromal follicular dendritic cells attracts CXCR5‐expressing cells towards the B‐cell follicles. Previous studies using a complex in vivo depletion model have suggested that CXCR5‐expressing conventional dendritic cells (cDC) help regulate the induction of Th2‐polarized responses. Here, transgenic mice with CXCR5 deficiency specifically restricted to CD11c⁺ cells were used to determine whether the specific absence CXCR5 on CD11c⁺ cells such as cDC would influence susceptibility to oral T. muris infection by affecting the Th1/Th2 balance. We show that in contrast to control mice, those which lacked CXCR5 expression on CD11c⁺ cells failed to clear T. muris infection and developed cytokine and antibody responses that suggested a disturbed Th1/Th2 balance with enhanced IFN‐γ expression. These data suggest an important role of CXCR5‐expressing CD11c⁺ cells such as cDC in immunity to oral T. muris infection.     

Antigen selection for future anti-Trichuris vaccines: a comparison of cytokine and antibody responses to larval and adult antigen in a primary infection

Trichuriasis, caused by the whipworm Trichuris trichiura , is endemic in tropical and subtropical areas, affecting approximately 1 billion people. Child anthelminthic treatment programmes are being implemented but repeated treatments are costly, may prevent the development of acquired immunity and can lead to the development of drug resistant parasites. Thus, the development of a vaccine which would lead to the acquisition of immunity at an earlier age and reduce community faecal egg output would be beneficial. Development of subunit vaccines requires the identification of protective antigens and their formulation in a suitable adjuvant. Trichuris muris is an antigenically similar laboratory model for T. trichiura. Subcutaneous vaccination with adult excretory–secretory products (ES) protects susceptible mouse strains from T. muris . Larval stages may contain novel and more relevant antigens which when incorporated in a vaccine induce worm expulsion earlier in infection than the adult worm products. This study finds negligible difference in the cellular and humoral immune response to T. muris adult and third stage larva(e) (L3) ES during a primary T. muris infection , but identifies high molecular weight proteins in both adult and L3 ES as potential vaccine candidates.     

The protective effect of a DNA vaccine encoding the Toxoplasma gondii cyclophilin gene in BALB/c mice

Toxoplasmosis is a world‐wide zoonosis that causes significant public health and veterinary problems. The study of vaccines remains the most promising method for the future prevention and control of toxoplasmosis. Recombinant Toxoplasma gondii cyclophilin has been shown to have potent PPIase and IL‐12‐inducing activities, thus promoting the stabilization of T. gondii's life cycle and maintaining the survival of its host during evolution. In this study, the T. gondii cyclophilin gene was used to construct a DNA vaccine (pVAX1‐TgCyP). The immune response and protective efficacy of the vaccine against T. gondii infection in BALB/c mice were evaluated. All BALB/c mice that were vaccinated with pVAX1‐TgCyP developed a high response with TgCyP‐specific antibodies, and significant splenocyte proliferation (P < 0·05) compared with pVAX1 vector and PBS groups. pVAX1‐TgCyP also induced a significant Th1 type immune response, indicated by the higher production of IL‐2 and IFN‐γ (P < 0·05). The survival rate of BALB/c mice increased significantly after vaccination with pVAX1‐TgCyP (37·5%) (P < 0·05). These results indicate that TgCyP is a highly efficacious vaccine candidate that can generate protective immunity against T. gondii infection in BALB/c mice.     

Protection and immunological study on two tetraspanin‐derived vaccine candidates against schistosomiasis japonicum

Tetraspanins (TSPs) are proteins found on the surface of helminth parasites of the genus Schistosoma and are regarded as potentially protective antigens. The large extracellular loop of Schistosoma mansoni tetraspanin‐2, Sm‐TSP‐2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. It is well recognized that CD4⁺ T‐cell‐dependent immunity might play an important role against schistosomes; however, the contribution of CD8⁺ T cells against multicellular pathogen is still uncertain. The exogenous protein‐pulsed dendritic cells (DCs) can easily activate CD4⁺ T cells response, while CD8⁺ T cells response was relatively difficult to be induced. In this study, we evaluated the immunogenicity of TSP2HD antigen (hydrophilic domain of the S. japonicum tetraspanin‐2) and TAT (the protein transduction domain of HIV‐1)‐coupled TSP2HD protein. As TAT‐fused protein could promote major histocompatibility complex class I‐dependent antigen presentation in vitro, TAT‐TSP2HD‐pulsed DCs induced stronger proliferation of schistosome‐specific CD8⁺ T cells compared with DCs incubated with TSP2HD alone. Vaccination with TAT‐TSP2HD‐pulsed DCs in vivo could improve disease outcome in S. japonicum‐infected mice and was slightly superior to vaccination with DCs treated with TSP2HD. In summary, these data showed that TAT fusion proteins could help activate CD8⁺ cells and Th1 cells and provide part protection against schistosome.     

Rice‐produced MSP142 of Plasmodium falciparum elicits antibodies that inhibit parasite growth in vitro

Many malaria antigens contain multiple disulphide bonds involved in the formation of inhibitory B‐cell epitopes. Producing properly folded malaria antigens in sufficient quantities for vaccination is often a challenge. The 42‐kDa fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1₄₂) is such a kind of malaria antigen. In this study, we investigated the expression of MSP1₄₂ in a rice system (9522, a cultivar of Oryza sativa ssp. japonica), which was used as a bioreactor for protein production. The MSP1₄₂ gene was synthesized according to rice‐preferred codons and transformed into rice plants via an Agrobacterium‐mediated method. The recombinant antigen was efficiently expressed in rice seeds with a level up to 1.56% of total soluble protein and was recognized by both the conformational monoclonal antibody 5.2 (mAb5.2) and the pooled sera of P. falciparum malaria patients. Rabbits were immunized intramuscularly with the purified MSP1₄₂ formulated with Freund's adjuvant. High antibody titres against MSP1₄₂ were elicited. The rabbit immune sera reacted well with the native protein of P. falciparum parasite and strongly inhibited the in vitro growth of blood‐stage P. falciparum parasites, demonstrating that transgenic rice can become an efficient bioreactor for the production of malaria vaccine antigens.     

Evaluation of immune responses induced by rhoptry protein 5 and rhoptry protein 7 DNA vaccines against Toxoplasma gondii

Infection with the protozoan parasite Toxoplasma gondii is widespread, and the organism can cause congenital infections in humans. The horizontal transmission of Toxoplasma is even more common than congenital. An effective vaccine strategy brings the prospect of improving Toxoplasma disease control. Rhoptry protein 5 (ROP5) and ROP7 are potential stimulators of humoral and cellular immune responses. In this study, we constructed a multi‐antigenic DNA vaccine expressing ROP5 and ROP7 of T. gondii and compared the protective efficacy to single‐gene vaccines and control groups. BALB/c mice were immunized intramuscularly three times. The levels of IgG antibodies and cytokines in mice immunized with the multi‐antigenic DNA vaccine (pROP5/ROP7) were significantly higher than those in the control mice. Mice vaccinated with pROP5/ROP7 showed a longer survival time (16 days) than single‐gene‐immunized mice (11 and 12 days, respectively) or control mice (8 days) after a challenge with 1 × 10⁴ tachyzoites of RH strain of T. gondii. Furthermore, after intragastric infection with 20 cysts of PRU strain of T. gondii, the number of brain cysts in mice immunized with pROP5/ROP7 was only 25% of the number in control mice. Our results showed that a DNA vaccine encoding ROP5 and ROP7 significantly enhanced protection against T. gondii challenge.     

Fusion of foreign T‐cell epitopes and addition of TLR agonists enhance immunity against Neospora caninum profilin in cattle

We demonstrated recently that immunization with recombinant Neospora caninum profilin (rNcPRO) induces limited protection and a regulatory T‐cell response in mice. The aim of this study was to evaluate the immune response elicited by rNcPRO in cattle and assess a strategy to enhance its immunogenicity, combining the addition of T‐cell epitopes and immune modulators. We developed a chimeric recombinant profilin fused to functional T‐cell epitopes present in the N‐terminal sequence of vesicular stomatitis virus (VSV) glycoprotein G (rNcPRO/G). Groups of three cattle were immunized with two doses (2 weeks apart) of rNcPRO or rNcPRO/G formulated with alum hydroxide or a nanoparticulated soya‐based adjuvant enriched with Toll‐like receptor (TLR) 2 and TLR9 agonists, aimed to tackle the MyD88 pathway (AVECplus). rNcPRO induced only a primary immune response (IgM mediated), while antibodies in rNcPRO/G‐vaccinated animals switched to IgG1 after the booster. The vaccine formulated with rNcPRO/G and AVECplus improved the production of systemic IFN‐γ and induced long‐term recall B‐cell responses. Overall, our study provides data supporting the use of T‐cell epitopes from VSV glycoprotein G and TLR agonists to enhance and modulate immunity to peptide antigens in bovines, particularly when using small proteins from parasites for which immune responses are usually feeble.     

Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA‐1

Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N‐terminal 65 kDa portion of Trypomastigote surface antigen‐1 (TSA‐1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA‐1 has not previously been addressed. To assess the variation in TSA‐1, we cloned and sequenced TSA‐1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA‐1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA‐A 02 allele suggests that interlineage variation in TSA‐1 would not impair the range and efficacy of a vaccine containing TSA‐1.     

Modulation of human dendritic cell activity by Giardia and helminth antigens

Giardia duodenalis is a common intestinal protozoan parasite known to modulate host immune responses, including dendritic cell (DC) function. Coinfections of intestinal pathogens are common, and thus, DCs may be concurrently exposed to antigens from multiple parasites. Here, we investigated the effects of G. duodenalis products on human monocyte‐derived DC function independently and in combination with helminth antigens (Ascaris suum and Trichuris suis). All antigens individually induced an anti‐inflammatory phenotype in DCs, reducing lipopolysaccharide (LPS)‐induced interleukin (IL)‐6, IL‐12p70 and tumour necrosis factor (TNF)‐α secretion. G. duodenalis and T. suis products also consistently upregulated IL‐10 production. Despite a similar modulation of cytokine secretion, additive effects between Giardia and helminth products were not observed, indicating a dominant effect of a single parasite stimulus and limited interactive effects on DC function. G. duodenalis trophozoites induced rapid apoptosis in DCs, which was not observed with the helminth antigens suggesting that the modulatory effects of G. duodenalis may override that of A. suum and T. suis. Thus, G. duodenalis modulates DC activity by modulating cytokine secretion and/or inducing apoptosis, which may be a parasite‐driven mechanism to dampen host immunity and establish chronic infections. The differential mechanisms of DC modulation by intestinal parasites warrant further attention.     

Cross‐protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis

Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania‐specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN‐γ, IL‐12 and GM‐CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti‐Leishmania Th1 response was maintained after infection, being the IFN‐γ production based mainly on CD4⁺ T cells. We described one conserved Leishmania‐specific protein that could compose a pan‐Leishmania vaccine.     

Evidence of cross‐stage CD8+ T cell epitopes in malaria pre‐erythrocytic and blood stage infections

Malaria parasites have a complex, multistage life cycle and there is a widely held view that each stage displays a distinct set of antigens presented to the immune system. Yet, molecular analysis of malaria parasites suggests that many putative antigenic targets are shared amongst the different stages. The specificities of these cross‐stage antigens and the functions of the immune responses they elicit are poorly characterized. It is well‐known that CD8+ T cells play opposing immune functions following Plasmodium berghei (Pb) infection of C57BL/6 mice. Whilst these cells play a crucial role in protective immunity against pre‐erythrocytic stages, they are implicated in the development of severe disease during blood stages. Recently, CD8+ T cell epitopes derived from proteins supposedly specific for either pre‐erythrocytic or blood stages have been described. In this brief report, we have compiled and confirmed data that the majority of the mRNAs and/or proteins from which these epitopes are derived display expression across pre‐erythrocytic and blood stages. Importantly, we provide evidence of cross‐stage immune recognition of the majority of these CD8+ T cell epitopes. Hence, our findings provide a resource to further examine the relevance of antigen‐specific cross‐stage responses during malaria infections.     

Naegleria fowleri immunization modifies lymphocytes and APC of nasal mucosa

We investigated whether intranasal immunization with amoebic lysates plus cholera toxin modified the populations of T and B lymphocytes, macrophages and dendritic cells by flow cytometry from nose‐associated lymphoid tissue (NALT), cervical lymph nodes (CN), nasal passages (NP) and spleen (SP). In all immunized groups, the percentage of CD4 was higher than CD8 cells. CD45 was increased in B cells from mice immunized. We observed IgA antibody‐forming cell (IgA‐AFC) response, mainly in NALT and NP. Macrophages from NP and CN expressed the highest levels of CD80 and CD86 in N. fowleri lysates with either CT or CT alone immunized mice, whereas dendritic cells expressed high levels of CD80 and CD86 in all compartment from immunized mice. These were lower than those expressed by macrophages. Only in SP from CT‐immunized mice, these costimulatory molecules were increased. These results suggest that N. fowleri and CT antigens are taking by APCs, and therefore, protective immunity depends on interactions between APCs and T cells from NP and CN. Consequently, CD4 cells stimulate the differentiation from B lymphocytes to AFC IgA‐positive; antibody that we previously found interacting with trophozoites in the nasal lumen avoiding the N. fowleri attachment to nasal epithelium.     

Composition of Caenorhabditis elegans extracellular vesicles suggests roles in metabolism,immunity, and aging

The nematode Caenorhabditis elegans has been instrumental in the identification of evolutionarily conserved mechanisms of aging. C. elegans also has recently been found to have evolutionarily conserved extracellular vesicle (EV) signaling pathways. We have been developing tools that allow for the detailed study of EV biology in C. elegans. Here we apply our recently published method for high specificity purification of EVs from C. elegans to carry out target-independent proteomic and RNA analysis of nematode EVs. We identify diverse coding and non-coding RNA and protein cargo types commonly found in human EVs. The EV cargo spectrum is distinct from whole worms, suggesting that protein and RNA cargos are actively recruited to EVs. Gene ontology analysis revealed C. elegans EVs are enriched for extracellular-associated and signaling proteins, and network analysis indicates enrichment for metabolic, immune, and basement membrane associated proteins. Tissue enrichment and gene expression analysis suggests the secreted EV proteins are likely to be derived from intestine, muscle, and excretory tissue. An unbiased comparison of the EV proteins with a large database of C. elegans genome-wide microarray data showed significant overlap with gene sets that are associated with aging and immunity. Taken together our data suggest C. elegans could be a promising in vivo model for studying the genetics and physiology of EVs in a variety of contexts including aging, metabolism, and immune response.

     

Effect of ES products from Anisakis (Nematoda: Anisakidae) on experimentally induced colitis in adult zebrafish

Inflammatory bowel disease (IBD) in developed countries is linked with elevated hygienic standards. One of the several factors involved in this question may be reduced exposure to the immunomodulatory effects of parasitic helminths. Several investigations on treatment of mice and humans with helminth‐derived substances have supported this notion, but underlying mechanisms remain unclear. This study therefore dissects to what extent a series of immune‐related genes are modulated in zebrafish with experimentally induced colitis following exposure to excretory‐secretory (ES) products isolated from larval Anisakis, a widely distributed fish nematode. Adult zebrafish intrarectally exposed to the colitis‐inducing agent TNBS developed severe colitis leading to 80% severe morbidity, but if co‐injected (ip) with Anisakis ES products, the morbidity rate was 50% at the end of the experiment (48 hours post‐exposure). Gene expression studies of TNBS‐treated zebrafish showed clear upregulation of a range of genes encoding inflammatory cytokines and effector molecules and some induction of genes related to the adaptive response. A distinct innate‐driven immune response was seen in both TNBS and TNBS + ES groups, but expression values were significantly depressed for several important pro‐inflammatory genes in the TNBS + ES group, indicating protective mechanisms of Anisakis ES compounds on intestinal immunopathology in zebrafish.     

Differential T‐cell responses to a chimeric Plasmodium falciparum antigen; UB05‐09, correlates with acquired immunity to malaria

The development of a sterilizing and cost‐effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05‐09 can serve as protective immunity markers by eliciting higher T‐cell responses in malaria semi‐immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05‐09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN‐γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN‐γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well‐conserved in nature. It is concluded that UB05, UB09 and the chimera UB05‐09 posses T‐cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated.     

Leishmania donovani infection drives the priming of human monocyte‐derived dendritic cells during Plasmodium falciparum co‐infections

Functional impairment of dendritic cells (DCs) is part of a survival strategy evolved by Leishmania and Plasmodium parasites to evade host immune responses. Here, the effects of co‐exposing human monocyte‐derived DCs to Leishmania donovani promastigotes and Plasmodium falciparum‐infected erythrocytes were investigated. Co‐stimulation resulted in a dual, dose‐dependent effect on DC differentiation which ranged from semi‐mature cells, secreting low interleukin(‐12p70 levels to a complete lack of phenotypic maturation in the presence of high parasite amounts. The effect was mainly triggered by the Leishmania parasites, as illustrated by their ability to induce semi‐mature, interleukin‐10‐producing DCs, that poorly responded to lipopolysaccharide stimulation. Conversely, P. falciparum blood‐stage forms failed to activate DCs and only slightly interfered with lipopolysaccharide effects. Stimulation with high L. donovani concentrations triggered phosphatidylserine translocation, whose onset presented after initiating the maturation impairment process. When added in combination, the two parasites could co‐localize in the same DCs, confirming that the leading effects of Leishmania over Plasmodium may not be due to mutual exclusion. Altogether, these results suggest that in the presence of visceral leishmaniasis–malaria co‐infections, Leishmania‐driven effects may overrule the more silent response elicited by P. falciparum, shaping host immunity towards a regulatory pattern and possibly delaying disease resolution.     

Immunity and immune modulation elicited by the larval cestode Mesocestoides vogae and its products

Larval cestodes (metacestodes) induce long‐lasting infections leading to considerable pathology in humans and livestock. Their survival is typically associated with Th2‐biased immune responses and immunosuppressive effects and depends on the parasite's ability to excrete/secrete antigens with immunoregulatory properties. Here, Mesocestoides vogae, a natural parasite of mice, is proposed as a new model species for the identification and characterization of cestode‐derived immunomodulatory factors. We followed the kinetics of immune parameters after infection with M. vogae or exposure to their excretory/secretory (ES) products in a permissive strain of mice. Besides, a dominant IL‐10 production and accumulation of macrophages and eosinophils expressing mRNA for Fizz‐1, YM1 and Arg‐1, mice showed minimal IFN‐γ and transient IL‐4 production at early time points with a complete loss at later stages of infection. We found that serum‐free ES products without host contamination directly induced M2 macrophages and suppressed IFN‐γ production in vivo and in vitro. This study highlights the use of the M. vogae as a cestode infection model and its ES products as a valuable tool for the identification of new therapeutic targets for the control of larval cestodiasis.     

Enhanced protection against Clonorchis sinensis induced by co‐infection with Trichinella spiralis in rats

Although co‐infection with multiple parasites is a frequent occurrence, changes in the humoral immune response against a pre‐existing parasite induced as a result of a subsequent parasitic infection remain undetermined. Here, we utilized enzyme‐linked immunosorbent assay (ELISA) to investigate antibody responses, cytokine production and enhanced resistance in Clonorchis sinensis‐infected rats (Sprague–Dawley) upon Trichinella spiralis infection. Higher levels of C. sinensis‐specific IgG and IgA were elicited upon T. spiralis infection, and these levels remained higher than in rats infected with C. sinensis alone. Upon subsequent infection with T. spiralis, IgG antibodies against C. sinensis appeared to be rapidly boosted at day 3, and IgA antibodies were boosted at day 7. Challenge infection of C. sinensis‐infected rats with T. spiralis induced substantial mucosal IgG and IgA responses in the liver and intestine and increases in antibody‐secreting plasma cells in the spleen and bone marrow. Subsequent infection also appeared to confer effective control of liver C. sinensis loads, resulting in enhanced resistance. Memory B cells generated in response to C. sinensis infection were rapidly amplified into antibody‐secreting cells upon T. spiralis infection. These results indicate that enhanced C. sinensis clearance induced by co‐infection is associated with systemic and mucosal IgG and IgA responses.