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.     

Extracellular vesicles and microvascular pathology: Decoding the active dialogue

Extracellular vesicles (EV) are a heterogeneous collection of membrane‐surrounded structures released from all studied cells, under both physiological and pathological conditions. These nano‐size vesicles carry complex cargoes including different classes of proteins, lipids and nucleic acids and are known to act as a communication and signalling vesicles in various cellular process. In addition to their role in development and progression of pathological disorders which make them potentially great biomarkers, EV have beneficial effects, as they take part in homeostasis. In this review we have analysed the evidence for the role of microvesicles and exosomes secreted from other cells on microvascular endothelium (EV uptake) as well as the role of endothelial‐derived vesicles on their neighbouring and distant cells (EV release).     

Extracellular vesicles from triple negative breast cancer promote pro-inflammatory macrophages associated with better clinical outcome

Tumor associated macrophages (TAMs), which differentiate from circulating monocytes, are pervasive across human cancers and comprise heterogeneous populations. The contribution of tumor-derived signals to TAM heterogeneity is not well understood. In particular, tumors release both soluble factors and extracellular vesicles (EVs), whose respective impact on TAM precursors may be different. Here, we show that triple negative breast cancer cells (TNBCs) release EVs and soluble molecules promoting monocyte differentiation toward distinct macrophage fates. EVs specifically promoted proinflammatory macrophages bearing an interferon response signature. The combination in TNBC EVs of surface CSF-1 promoting survival and cargoes promoting cGAS/STING or other activation pathways led to differentiation of this particular macrophage subset. Notably, macrophages expressing the EV-induced signature were found among patients’ TAMs. Furthermore, higher expression of this signature was associated with T cell infiltration and extended patient survival. Together, this data indicates that TNBC-released CSF-1-bearing EVs promote a tumor immune microenvironment associated with a better prognosis in TNBC patients.

Tumors are infiltrated by different populations of immune cells, macrophages, in particular, being major components of the tumor microenvironment. Tumor-associated macrophages (TAMs) favor tumor progression, promoting cancer cell invasion and metastasis in mouse models (1). Recent single-cell analyses of human cancers revealed the heterogeneity of macrophage populations, thus challenging our understanding of TAM biology (2–4). TAMs derive from circulating monocytes that are recruited into the tumor via the CCL2-CCR2 chemokine signaling pathway (5, 6). The fate of monocytes is not predetermined and largely depend on the microenvironmental cues they encounter (7). Specifically, the identity of tumor-derived factors that contribute to TAM heterogeneity and the mechanisms underlying intratumoral monocyte differentiation remain unclear.Among the signals that can impact TAM differentiation and activation, cytokines and chemokines are well-characterized factors (8); extracellular vesicles (EVs), however, represent novel candidates. EVs are complex vehicles of intercellular communication and were suggested to have an impact on macrophage activation (9–12). EVs, such as exosomes, ectosomes, microvesicles, and oncosomes, are membrane-enclosed structures that contain proteins and nucleic acids and can be released into the extracellular environment by all cell types, including cancer cells (13, 14). Once released, EVs can interact with recipient cells and modulate their function (15). Particularly, EVs released by cancer cells play an important role in shaping the tumor immune microenvironment. EVs were shown to modulate lymphocytes and myeloid cell functions in cancer by triggering either protumor or anti-tumor immune responses (16, 17), which may depend on numerous factors, such as the cancer type, stage, or EV subtype analyzed (18). Here, we address the specific contribution of tumor-derived EVs, as compared to the tumor-derived soluble factors, in driving TAM heterogeneity. For this, we focus on human breast cancer since these tumors are highly infiltrated with macrophages (1). Our results confirm a striking functional difference of EVs and soluble factors in tuning TAM profile that we attribute to the combination of survival and activation signals carried by EVs. Furthermore, we unravel an unexpected ability of TNBC-EVs to promote an inflammatory tumor microenvironment associated with a better clinical outcome.     

Bulk immunoassays for analysis of extracellular vesicles

There is increasing clinical interest in extracellular vesicles (EV) for diagnostic and treatment purposes. This review provides an overview of bulk immunoassays to analyse EV. Western blot and enzyme-linked immunosorbent assay are still the two predominant bulk immunoassays. Recently, new assays have become available that can detect exposure to EV concentrations that are up to 10,000-fold lower. This is advantageous for applications that detect rare EV. Other important parameters are the detectable concentration range, the required sample volume, whether simultaneous presence of different antigens on a single EV can be detected, size selectivity of each assay and practical considerations. In this review, we will explain the working principles of the traditional and novel assays together with their performance parameters. The most sensitive assays are micro-nuclear magnetic resonance, surface plasmon resonance, and time-resolved fluorescent immunoassay.     

Vaccination with recombinant actin from scab mites and evaluation of its protective efficacy against Psoroptes cuniculi infection

The mite Psoroptes cuniculi is globally widespread and has a serious impact on commercial rabbit breeding. Current treatment methods are based on chemotherapy. Because of the disadvantages of these methods, alternative measures are required, and vaccination is one of the most promising strategies. Here, we cloned and expressed the recombinant P. cuniculi actin gene (rPc‐act). Antiserum levels against rPc‐act in rabbits were used to locate actin distribution in mite sections. Challenge trials were carried out to evaluate the immunity protection of rPc‐act in rabbits, with antibody levels determined by ELISA. Sequence analysis of this gene fragment showed 89·26% and 84·91% identity to Sarcoptes scabiei and Mayetiola destructor sequences, respectively. Immunohistochemistry showed rPc‐act to locate widely throughout the mites, especially in feet and muscle tissues. Recombinant P. cuniculi actin with QuliA adjuvant was used to immunize six rabbits. Each animal was challenge‐infested with 25–50 adult mites. Although IgE levels showed no significant difference to controls, IgG levels were significantly higher, and clinical development showed no significantly different severity of lesions in vaccinated rabbits than in the controls. This study showed that rPc‐act is a muscular isotype actin and has no clinical protective efficacy against P. cuniculi.     

The Significance of Phenotyping and Quantification of Plasma Extracellular Vesicles Levels Using High-Sensitivity Flow Cytometry during COVID-19 Treatment

New investigation results point to the potential participation of extracellular vesicles (EVs) in the pathogenesis of coronavirus infection, its progression, and mechanisms of the therapy effectiveness. This dictates the necessity to transfer scientific testing technologies to medical practice. Here, we demonstrated the method of phenotyping and quantitative analysis of plasma EVs based on differential centrifugation, immunostaining, and high-sensitivity multicolor flow cytometry. We used EV markers that were potentially associated with SARS-CoV-2 dissemination via vesicles and cell-origination markers, characterizing objects from different cell types that could influence clinical manifestation of COVID-19. Plasma levels of CD235a+ and CD14+ EVs in patients with moderate infection were significantly increased while CD8+ and CD19+ EVs were decreased comparing with HD. Patients with severe infection had lower levels of CD4+, CD19+, and CD146+ EVs than HD. These findings demonstrate that EV concentrations in COVID-19 are severity related. Moreover, the three-point dynamic assessment demonstrated significant loss of CD63+ and CD147+ plasma EVs. The used method can be a convenient tool for vital infection pathogenesis investigation and for COVID-19 diagnostics.     

On the Relationship of Viral Particles and Extracellular Vesicles: Implications for Viral Vector Technology

Gene therapy vectors derived from different viral species have become a fixture in biomedicine, both for direct therapeutic intervention and as tools to facilitate cell-based therapies, such as chimeric antigen receptor-based immunotherapies. On the contrary, extracellular vesicles have only recently gained a massive increase in interest and, concomitantly, knowledge in the field has drastically risen. Viral infections and extracellular vesicle biology overlap in many ways, both with pro- and antiviral outcomes. In this review, we take a closer look at these interactions for the most prominent groups of viral vectors (Adenoviral, Adeno-associated and Retro/Lentiviral vectors) and the possible implications of these overlaps for viral vector technology and its biomedical applications.     

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.     

A multivalent chimeric vaccine composed of Schistosoma mansoni SmTSP‐2 and Sm29 was able to induce protection against infection in mice

Schistosoma mansoni is a blood fluke parasite responsible for schistosomiasis. The best long‐term strategy to control schistosomiasis is through immunization combined with drug treatment. In this study, we cloned, expressed and purified SmTSP‐2 fused to the N‐ and C‐terminal halves of Sm29 and tested these chimeras as vaccine candidates using an adjuvant approved to be used in humans. The results demonstrated that vaccination with SmTSP‐2 fused to N‐ or C‐terminus of Sm29‐induced reduction in worm burden and liver pathology when compared to control animals. Additionally, we detected high levels of mouse‐specific IgG, IgG1 and IgG2a against both chimeras and significant amounts of IFN‐γ and TNF‐α and no IL‐4. Finally, studies with sera from patients resistant to infection and living in schistosomiasis endemic areas revealed high levels of specific IgG to both chimeras when compared to healthy individuals. In conclusion, SmTSP‐2/Sm29 chimeras tested here induced partial protection against infection and might be a potential vaccine candidate.     

Serology and longevity of immunity against Echinococcus granulosus in sheep and llama induced by an oil‐based EG95 vaccine

An oil‐based formulation of the EG95 vaccine to protect grazing animals against infection with Echinococcus granulosus was formulated in Argentina. The efficacy of the vaccine was monitored by serology in sheep and llama (Lama glama) and was compared to the serology in sheep previously published using a QuilA‐adjuvanted vaccine. Long‐term efficacy was also tested in sheep by challenging with E. granulosus eggs of the G1 strain 4 years after the beginning of the trial. The serological results for both sheep and llama were similar to those described previously, except that there was a more rapid response after the first vaccination. A third vaccination given after 1 year resulted in a transient boost in serology that lasted for about 12 months, which was similar to results previously described. Sheep challenged after 4 years with three vaccinations presented 84·2% reduction of live cysts counts compared with control group, and after a fourth vaccination prior to challenge, this reduction was 94·7%. The oil‐based vaccine appeared to be bio‐equivalent to the QuilA vaccine.     

Enhanced acquired antibodies to a chimeric Plasmodium falciparum antigen; UB05‐09 is associated with protective immunity against malaria

It has been shown that covalently linking two antigens could enhance the immunogenicity of the chimeric construct. To prioritize such a chimera for malaria vaccine development, it is necessary to demonstrate that naturally acquired antibodies against the chimera are associated with protection from malaria. Here, we probe the ability of a chimeric construct of UB05 and UB09 antigens (UB05‐09) to better differentiate between acquired immune protection and susceptibility to malaria. In a cross‐sectional study, recombinant UB05‐09 chimera and the constituent antigens were used to probe for specific antibodies in the plasma from children and adults resident in a malaria‐endemic zone, using the enzyme‐linked immunosorbent assay (ELISA). Anti‐UB05‐09 antibody levels doubled that of its constituent antigens, UB09 and UB05, and this correlated with protection against malaria. The presence of enhanced UB05‐09‐specific antibody correlated with the absence of fever and parasitaemia, which are the main symptoms of malaria infection. The chimera is more effective in detecting and distinguishing acquired protective immunity against malaria than any of its constituents taken alone. Online B‐cell epitope prediction tools confirmed the presence of B‐cell epitopes in the study antigens. UB05‐09 chimera is a marker of protective immunity against malaria that needs to be studied further.     

Extracellular vesicles and melatonin benefit embryonic develop by regulating reactive oxygen species and 5-methylcytosine

Embryo culture conditions are crucial as they can affect embryo quality and even offspring. Oviductal extracellular vesicles (EVs) long been considered a major factor influencing interactions between the oviduct and embryos, and thus its absence is associated with inferior embryonic development in in vitro culture. Herein, we demonstrated that melatonin is present in oviduct fluids and oviduct fluid-derived EVs. Addition of either EVs (1.87 × 10¹¹ particles/mL) or melatonin (340 ng/mL) led to a significant downregulation of reactive oxygen species (ROS) and 5-methylcytosine (5-mC), as well as an increase in the blastocyst rate of embryos, which was inhibited by the addition of luzindole—a melatonin receptor agonist. A combination of EVs (1.87 × 10¹⁰ particles/mL) and melatonin (at 34.3 pg/mL) led to the same results as well as a significant decrease in the apoptosis index and increase in the inner cell mass (ICM)/trophectoderm (TE) index. These results suggest that an EV-melatonin treatment benefits embryonic development. Our findings provide insights into the role of EVs and melatonin during cell communication and provide new evidence of the communication between embryos and maternal oviduct.     

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.     

Evaluation of a novel lentiviral vaccine expressing KMP11‐HASPB fusion protein against Leishmania infantum in BALB/c mice

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania protein against which antibodies are produced in the sera of cutaneous and visceral Leishmaniasis (VL) patients. Kinetoplastid membrane protein 11 (KMP11) is another protein antigen of Leishmania which is reported as a promising candidate for vaccination of VL. It is a highly conserved surface protein present in all members of kinetoplastid family and is expressed in both promastigotes and amastigotes. In this study, the coding sequence of KMP11 and HASPB was cloned into a pCDH‐cGFP lentiviral vector as a fusion protein. The gene expression was confirmed using RT‐PCR and Western blot methods. After injection of the recombinant KMP11‐HASPB‐expressing lentiviruses to BALB/c mice, using ELISA technique, a significant increase in IFN‐γ and IL‐4 as well as IgG1 and IgG2a was observed compared to the control group. Furthermore, the number of parasites in the liver and spleen of vaccinated mice decreased significantly compared with the control group.     

Maternal immunization promotes the immune response of neonates towards hepatitis B vaccine

Infants infected with hepatitis B virus (HBV) face the risk of developing severe complications. Unfortunately, in spite of universal vaccination programmes, 5% or more of vaccinated newborns still do not achieve protective levels of anti‐hepatitis B virus surface antigen titres (anti‐HBs). The aim of this study was to use animal experiments and population‐based research to determine whether maternal vaccination against HBV affects the outcome of neonatal vaccination. Six sows and 53 newborn piglets were used for this study and randomly assigned to the vaccination group (three 20 μg doses of recombinant HBV vaccine). All the piglets were followed up to 10 weeks of age, and peripheral blood was withdrawn for measurement of anti‐HBs. A cross‐sectional study was also conducted on 449 mothers with infants. A structured questionnaire was used to collect demographic, medical and maternal data, and their peripheral blood was collected for measurement of anti‐HBs. The results of animal experiments demonstrated that nonvaccinated piglets born to vaccinated sows and nonvaccinated piglets born to nonvaccinated sows were negative for anti‐HBs. Repeated measures analysis of variance showed that the titres of anti‐HBs in vaccinated piglets born to vaccinated sows were significantly higher than in vaccinated piglets born to nonvaccinated sows (P < 0.05). In a population‐based study, a cumulative logistic regression analysis showed that the strongest influences on neonatal anti‐HBs titres were delay of the first vaccination dose [OR = 3.02(95% CI: 1.72–5.30)] and maternal anti‐HBs titres [OR = 2.48(95% CI: 2.03–3.04)]. In conclusion, high maternal anti‐HBs titres can enhance the response to HBV vaccination in infants.