With Chitosan and PLGA as the Delivery Vehicle, Toxoplasma gondii Oxidoreductase-Based DNA Vaccines Decrease Parasite Burdens in Mice.

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan that can cause serious public health problems. However, there is no effectively preventive or therapeutic strategy available for human and animals. In the present study, we developed a DNA vaccine encoding T. gondii oxidoreductase from short-chain dehydrogenase/reductase family (TgSDRO-pVAX1) and then entrapped in chitosan and poly lactic-co-glycolic acid (PLGA) to improve the efficacy. When encapsulated in chitosan (TgSDRO-pVAX1/CS nanospheres) and PLGA (TgSDRO-pVAX1/PLGA nanospheres), adequate plasmids were loaded and released stably. Before animal immunizations, the DNA vaccine was transfected into HEK 293-T cells and examined by western blotting and laser confocal microscopy. Th1/Th2 cellular and humoral immunity was induced in immunized mice, accompanied by modulated secretion of antibodies and cytokines, promoted the maturation and MHC expression of dendritic cells, and enhanced the percentages of CD4+ and CD8+ T lymphocytes. Immunization with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres conferred significant immunity with lower parasite burden in the mice model of acute toxoplasmosis. Furthermore, our results also lent credit to the idea that TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres are substitutes for each other. In general, the current study proposed that TgSDRO-pVAX1 with chitosan or PLGA as the delivery vehicle is a promising vaccine candidate against acute toxoplasmosis.

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis.

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.

Rescue of maternal immune activation-induced behavioral abnormalities in adult mouse offspring by pathogen-activated maternal Treg cells.

Maternal immune activation (MIA) induced by lipopolysaccharides or polyinosinic:polycytidylic acid injections can induce behavioral abnormalities in adult mouse offspring. Here, we used the soluble tachyzoite antigen from Toxoplasma gondii, a parasite that infects approximately two billion people, to induce MIA in mice. The adult male offspring showed autism-relevant behaviors and abnormal brain microstructure, along with a pro-inflammatory T-cell immune profile in the periphery and upregulation of interleukin-6 in brain astrocytes. We show that adoptive transfer of regulatory T (Treg) cells largely reversed these MIA-induced phenotypes. Notably, pathogen-activated maternal Treg cells showed greater rescue efficacy than those from control donors. Single-cell RNA sequencing identified and characterized a unique group of pathogen-activated Treg cells that constitute 32.6% of the pathogen-activated maternal Treg population. Our study establishes a new preclinical parasite-mimicking MIA model and suggests therapeutic potential of adoptive Treg cell transfer in neuropsychiatric disorders associated with immune alterations.

Immune induction by adjuvanted Leishmania donovani vaccines against the visceral leishmaniasis in BALB/c mice.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by Leishmania donovani or Leishmania infantum. Currently, the patients are treated with chemotherapeutic drugs; however, their toxicity limits their use. It would be desirable to develop a vaccine against this infection. In this study, we assessed the efficacy of different vaccine formulations at variable time points. Heat-killed (HK) antigen of Leishmania donovani was adjuvanted with two adjuvants (AddaVax and Montanide ISA 201) and three immunizations at a gap of 2 weeks (wk) were given to BALB/c mice. After 2 weeks of the last booster, mice were given challenge infection and sacrificed before challenge and after 4wk, 8wk, and 12 wk post-challenge. Significant protective immunity was observed in all the immunized animals and it was indicated by the notable rise in delayed-type hypersensitivity (DTH) response, remarkably declined parasite burden, a significant increase in the levels of interferon-gamma (IFN-γ), interleukin-12, interleukin-17 (Th1 cytokines), and IgG2a in contrast to infected control mice. Montanide ISA 201 with HK antigen provided maximum protection followed by AddaVax with HK and then HK alone. These findings elaborate on the importance of the tested adjuvants in the vaccine formulations against murine visceral leishmaniasis.

Pfs230 yields higher malaria transmission-blocking vaccine activity than Pfs25 in humans but not mice.

BACKGROUNDVaccines that block human-to-mosquito Plasmodium transmission are needed for malaria eradication, and clinical trials have targeted zygote antigen Pfs25 for decades. We reported that a Pfs25 protein-protein conjugate vaccine formulated in alum adjuvant induced serum functional activity in both US and Malian adults. However, antibody levels declined rapidly, and transmission-reducing activity required 4 vaccine doses. Functional immunogenicity and durability must be improved before advancing transmission-blocking vaccines further in clinical development. We hypothesized that the prefertilization protein Pfs230 alone or in combination with Pfs25 would improve functional activity.METHODSTransmission-blocking vaccine candidates based on gamete antigen Pfs230 or Pfs25 were conjugated with Exoprotein A, formulated in Alhydrogel, and administered to mice, rhesus macaques, and humans. Antibody levels were measured by ELISA and transmission-reducing activity was assessed by the standard membrane feeding assay.RESULTSPfs25-EPA/Alhydrogel and Pfs230D1-EPA/Alhydrogel induced similar serum functional activity in mice, but Pfs230D1-EPA induced significantly greater activity in rhesus monkeys that was enhanced by complement. In US adults, 2 vaccine doses induced complement-dependent activity in 4 of 5 Pfs230D1-EPA/Alhydrogel recipients but no significant activity in 5 Pfs25-EPA recipients, and combination with Pfs25-EPA did not increase activity over Pfs230D1-EPA alone.CONCLUSIONThe complement-dependent functional immunogenicity of Pfs230D1-EPA represents a significant improvement over Pfs25-EPA in this comparative study. The rhesus model is more predictive of the functional human immune response to Pfs230D1 than is the mouse model.TRIAL REGISTRATIONClinicalTrials.gov NCT02334462.FUNDINGIntramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

A Phospholipase-A Activity in Soluble Leishmania Antigens Causes Instability of Liposomes.

AIM: This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. OBJECTIVES: Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). METHODS: Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). RESULT: The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. CONCLUSION: Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.

Evaluation of the protective efficacy of a Leishmania protein associated with distinct adjuvants against visceral leishmaniasis and in vitro immunogenicity in human cells.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.

A TLR9-adjuvanted vaccine formulated into dissolvable microneedle patches or cationic liposomes protects against leishmaniasis after skin or subcutaneous immunization.

Re-emergence and geographic expansion of leishmaniasis is accelerating efforts to develop a safe and effective Leshmania vaccine. Vaccines using Leishmania recombinant antigens, such as LiHyp1, which is mostly present in the amastigote parasite form, are being developed as a next generation to crude killed parasite-based vaccines. The main objective of this work was to develop a LiHyp1-based vaccine and determine if it can induce protective immunity in BALB/c mice when administered using a dissolvable microneedle (DMN) patch by the skin route. The LiHyp1 antigen was incorporated into cationic liposomes (CL), with or without the TLR9 agonist, CpG. The LiHyp1-liposomal vaccines were characterized with respect to size, protein encapsulation rates and retention of their physical characteristics after incorporation into the DMN patch. DMN mechanical strength and skin penetration ability were tested. A vaccine composed of LiHyp1, CpG and liposomes and subcutaneously injected or a vaccine containing antigen and CpG in DMN patches, without liposomes, induced high antibody responses and significant levels of protection against L. donovani parasite infection. This study progresses the development of an efficacious leishmania vaccine by detailing promising vaccine formulations and skin delivery technologies and it addresses protective efficacy of a liposome-based dissolvable microneedle patch vaccine system.

In vitro evaluations on canine monocyte-derived dendritic cells of a nanoparticles delivery system for vaccine antigen against Echinococcus granulosus.

Since dogs play a central role in the contamination of humans and livestock with Echinococcus granulosus, the development of an effective vaccine for dogs is essential to control the disease caused by this parasite. For this purpose, a formulation based on biodegradable polymeric nanoparticles (NPs) as delivery system of recombinant Echinococcus granulosus antigen (tropomyosin EgTrp) adjuved with monophosphoryl lipid A (MPLA) has been developed. The obtained nanoparticles had a size of approximately 200 nm in diameter into which the antigen was correctly preserved and encapsulated. The efficiency of this system to deliver the antigen was evaluated in vitro on canine monocyte-derived dendritic cells (cMoDCs) generated from peripheral blood monocytes. After 48 h of contact between the formulations and cMoDCs, we observed no toxic effect on the cells but a strong internalization of the NPs, probably through different pathways depending on the presence or not of MPLA. An evaluation of cMoDCs activation by flow cytometry showed a stronger expression of CD80, CD86, CD40 and MHCII by cells treated with any of the tested formulations or with LPS (positive control) in comparison to cells treated with PBS (negative control). A higher activation was observed for cells challenged with EgTrp-NPs-MPLA compared to EgTrp alone. Formulations with MPLA, even at low ratio of MPLA, give better results than formulations without MPLA, proving the importance of the adjuvant in the nanoparticles structure. Moreover, autologous T CD4+ cell proliferation observed in presence of cMoDCs challenged with EgTrp-NPs-MPLA was higher than those observed after challenged with EgTrp alone (p<0.05). These first results suggest that our formulation could be used as an antigen delivery system to targeting canine dendritic cells in the course of Echinococcus granulosus vaccine development.

Toxoplasma gondii RPL40 is a circulating antigen with immune protection effect.

Screening and identification of protective antigens are essential for the prevention of infections with Toxoplasma gondii (Nicolle et Manceaux, 1908). In our previous study, T. gondii ribosomal-ubiquitin protein L40 (TgRPL40) was identified as a circulating antigen. However, the function and protective value of TgRPL40 was unknown. In the current study, recombinant TgRPL40 was expressed in Escherichia coli BL21 and antibody was prepared. Western blotting analysis indicated that TgRPL40 was present in circulating antigens and excretory/secretary antigens (ESA). Immunofluorescence and immunoelectron microscopy analysis revealed that TgRPL40 protein is widely distributed in the tachyzoites. Immunisation with recombinant TgRPL40 prolonged the survival of mice infected with tachyzoites. Quantitative real-time polymerase chain reaction analysis showed that immunisation with recombinant TgRPL40 reduced the parasite burden in blood, liver, spleen and brain of mice infected with tachyzoites. These observations indicate that TgRPL40 is a circulating antigen and is an effector of immune protection against acute T. gondii infection.

Co-administration of chitin micro-particle and Leishmania antigen proposed a new immune adjuvant against experimental leishmaniasis.

AIMS: We investigated the protective effect of chitin micro-particle (CMP) as an adjuvant against Leishmania infection in BALB/c mice. METHODS: Mice were immunized subcutaneously with soluble Leishmania antigen (SLA) plus CMP (100 µg SLA + 100 µg CMP/100 µL) as the test group. Three weeks after the last immunization, test and control groups were infected by Leishmania major (L major). Eight weeks post-infection, evaluation of parasites load in lymph nodes was performed using limiting dilution assay. Then, the spleen cell cytokine response (TNF-α, IFN-γ, IL-4, IL-10, IL-17 and IL-27) to SLA among vaccinated and nonvaccinated groups was investigated using ELISA. Serum levels of IgG1 and IgG2a were measured as well. RESULTS: The SLA plus CMP group demonstrated the protection. The responses included reduced lesion formation and lower parasite load. Also, in comparison with control group higher levels of IFN-γ and, IL-10 in the culture of spleen cells, and lower levels of IgG1 in sera were seen in SLA plus CMP group. CONCLUSION: The data supported the possibility of using CMP as a suitable adjuvant in Leishmania vaccination.

Candidate antigenic epitopes for vaccination and diagnosis strategies of Toxoplasma gondii infection: A review.

Toxoplasmosis caused by an obligatory intracellular protozoan parasite of Toxoplasma gondii threats a wide spectrum of human and animal hosts. It has been shown that the intensity of the disease in humans depends on the host's immune responses. Immunological investigations on whole protein molecules of T. gondii have shown that these antigens are not fully responsible for the immune response, which leads to a decrease in specificity and affinity of the antigen (epitope)-antibody (paratope) binding. Currently, epitopes have shown promising entities to stimulate B, T, cytotoxic T lymphocyte, and NK cells resulting in enhancement of protective immunity against toxoplasmosis patients. Thus, the accurate designing, prediction, and conducting of antigenic epitopes of T. gondii (with linear and/or spatial structures (can augment our understanding about development of new serological diagnostic kits and vaccines. The current review provides an update on the latest advances of current epitopes described against toxoplasmosis including B cell/T cell epitopes, antigen types, parasite strains, epitope sequences, assay settings (in vitro and/or in vivo), and target strategy. Present results disclosed that the designing of effective multiepitopes of T. gondii by in silico modeling and immunoinformatics tools can strengthen our knowledge about triggering of epitope-based vaccine/diagnosis strategies in future perspectives.

An immunoinformatics approach for design and validation of multi-subunit vaccine against Cryptosporidium parvum.

An immunoinformatics-based approach is explored for potential multi-subunit vaccine candidates against Cryptosporidium parvum. We performed protein structure based systematic methodology for the development of a proficient multi-subunit vaccine candidate against C. parvum based on their probability of antigenicity, allergenicity and transmembrane helices as the screening criteria. The best-screened epitopes like B-cell epitopes (BCL), Helper T-lymphocytes (HTL) and cytotoxic T- lymphocytes (CTL) were joined by using the appropriate linkers to intensify and develop the presentation and processing of the antigenic molecules. Modeller software was used to generate the best 3D model of the subunit protein. RAMPAGE and other web servers were employed for the validation of the modeled protein. Furthermore, the predicted modeled structure was docked with the two known receptors like TLR2 and TLR4 through ClusPro web server. Based on the docking score, the multi-subunit vaccine docked with TLR2 was subjected to energy minimization by molecular dynamics (MD) simulation to examine their stability within a solvent system. From the simulation study, we found that the residue Glu-107 of subunit vaccine formed a hydrogen bond interaction with Arg-299 of the TLR2 receptor throughout the time frame of the MD simulation. The overall results showed that the multi-subunit vaccine could be an efficient vaccine candidate against C. parvum.

Evaluation of immune protection against Toxoplasma gondii infection in mice induced by a multi-antigenic DNA vaccine containing TgGRA24, TgGRA25 and TgMIC6.

Toxoplasma gondii infection is prevalent in humans and animals worldwide. In this study, recombinant eukaryotic expression plasmids (pVAX-GRA24, pVAX-GRA25 and pVAX-MIC6) were constructed, and then injected into Kunming mice intramuscularly, as cocktailed plasmids or as single-gene plasmids. We evaluated immune protective responses by detecting the titer of antibodies and cytokine production of IFN-γ, IL-2, IL-4, IL-10, IL-12 and IL-23, the percentages of the subclasses of T lymphocytes, as well as the records of the survival time and cyst decrement in the brain of the mouse model after challenge with the T. gondii RH and Pru strains, respectively. Compared with the control groups, antibody and cytokine production were significantly increased, while the survival times of mice in all immunized groups were also prolonged, and the number of T. gondii cysts in their brains were decreased significantly (29.03% for pVAX-GRA24; 40.88% for pVAX-GRA25; 37.70% for pVAX-MIC6; 48.06% for pVAX-GRA24 + pVAX-GRA25; and 55.37% for pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6). The mouse group immunized with the three-gene cocktail (TgGRA24 + TgGRA25 + TgMIC6) had better performance in each detection index than the mouse groups immunized with the two-gene cocktail of TgGRA24 + TgGRA25, which was better than that in the group immunized with the single gene vaccine of TgGRA24, TgMIC6 or TgGRA25. In conclusion, TgGRA24 or TgGRA25 may be good vaccine candidates against T. gondii infection, but the three-gene cocktail of TgGRA24, TgMIC6 and TgGRA25 may induce the strongest protective immunity. Further studies of multi-antigenic DNA vaccines or cocktailed vaccines against T. gondii infection are necessary.

TITLE: Évaluation de la protection immunitaire contre l'infection par Toxoplasma gondii chez la souris, induite par un vaccin à ADN multi-antigénique contenant TgGRA24, TgGRA25 et TgMIC6. ABSTRACT: L'infection à Toxoplasma gondii est répandue chez les humains et les animaux dans le monde entier. Dans cette étude, des plasmides d'expression eucaryotes recombinants (pVAX-GRA24, pVAX-GRA25 et pVAX-MIC6) ont été construits, puis injectés à des souris Kunming par voie intramusculaire, comme cocktails de plasmides ou comme plasmides à un seul gène. Nous avons évalué les réponses de protection immunitaire en détectant le titre des anticorps et la production de cytokines IFN-γ, IL-2, IL-4, IL-10, IL-12 et IL-23, les pourcentages des sous-classes des lymphocytes T, ainsi qu'en mesurant les temps de survie et de décrément des kystes dans le cerveau du modèle souris après challenge par les souches RH et Pru de T. gondii, respectivement. Comparativement aux groupes témoins, la production d'anticorps et de cytokines était significativement accrue, tandis que le temps de survie des souris de tous les groupes immunisés était également prolongé et que le nombre de kystes de T. gondii dans leur cerveau diminuait de manière significative (29,03 % pour pVAX-GRA24 ; 40,88 % pour pVAX-GRA25 ; 37,70 % pour pVAX-MIC6 ; 48,06 % pour pVAX-GRA24 + pVAX-GRA25 ; 55,37 % pour pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6). Le groupe de souris immunisées avec les cocktails à trois gènes (TgGRA24 + TgGRA25 + TgMIC6) présentait la meilleure performance dans chaque indice de détection par rapport aux groupes de souris immunisées avec des cocktails à deux gènes de TgGRA24 + TgGRA25, qui était supérieur à ceux immunisés avec les vaccins monogéniques TgGRA24, TgMIC6 ou TgGRA25. En conclusion, TgGRA24 ou TgGRA25 peuvent être de bons candidats au vaccin contre l'infection à T. gondii, mais un cocktail à trois gènes de TgGRA24, TgMIC6 et TgGRA25 peut induire la plus forte immunité protectrice. Des études supplémentaires sur les vaccins à ADN multi-antigéniques ou les vaccins en cocktail contre l'infection à T. gondii sont nécessaires.

Pam3CSK4 adjuvant given intranasally boosts anti-Leishmania immunogenicity but not protective immune responses conferred by LaAg vaccine against visceral leishmaniasis.

The use of adjuvants in vaccine formulations is a well-established practice to improve immunogenicity and protective immunity against diseases. Previously, we have demonstrated the feasibility of intranasal vaccination with the antigen of killed Leishmania amazonensis promastigotes (LaAg) against experimental leishmaniasis. In this work, we sought to optimize the immunogenic effect and protective immunity against murine visceral leishmaniasis conferred by intranasal delivery of LaAg in combination with a synthetic TLR1/TLR2 agonist (Pam3CSK4). Intranasal vaccination with LaAg/PAM did not show toxicity or adverse effects, induced the increase of delayed-type hypersensitivity response and the production of inflammatory cytokines after parasite antigen recall. However, mice vaccinated with LaAg/PAM and challenged with Leishmania infantum presented significant reduction of parasite burden in both liver and spleen, similar to those vaccinated with LaAg. Although LaAg/PAM intranasal vaccination had induced higher frequencies of specific CD4+ and CD8+ T cells and increased levels of IgG2a antibody isotype in serum, both LaAg and LaAg/PAM groups presented similar levels of IL-4 and IFN-y and decreased production of IL-10 when compared to controls. Our results provide the first evidence of the feasibility of intranasal immunization with antigens of killed Leishmania in association with a TLR agonist, which may be explored for developing an effective and alternative strategy for vaccination against visceral leishmaniasis.

Parasite load stemming from immunization route determines the duration of liver-stage immunity.

Immunization with radiation-attenuated Plasmodium sporozoites (RAS) induces sterile and long-lasting protective immunity. Although intravenous (IV) route of RAS immunization is reported to induce superior immunity compared to intradermal (ID) injection, its role in the maintenance of sterile immunity is yet to be understood. We investigated whether the route of homologous sporozoite challenge of Plasmodium berghei (Pb) RAS-immunized mice would influence the longevity of protection. C57BL/6 mice immunized with Pb-RAS by IV were 100% protected upon primary IV/ID sporozoite challenge. In contrast, ID immunization resulted in 80% protection, regardless of primary challenge route. Interestingly, the route of primary challenge was found to bring difference in the maintenance of sterile protection. While IV Pb RAS-immunized mice remained protected at all challenges regardless of the route of primary challenge, ID Pb-RAS-immunized mice receiving ID primary challenge became parasitaemic upon secondary IV challenge. Significantly, primary IV challenge of Pb RAS ID-immunized mice resulted in 80% and 50% survival at secondary and tertiary challenges, respectively. According to phenotypically diverse liver CD8+ T cells, the percentages and the numbers of both CD8+ T effector memory and resident memory cells were significantly higher in IV than in ID Pb RAS-immunized mice. IFN-γ-producing CD8+ T cells specific to Pb TRAP130 and MIP-4-Kb-17 were also found significantly higher in IV mice than in ID mice. The enhanced T-cell generation and the longevity of protection appear to be dependent on the parasite load during challenge when infection is tolerated under suboptimal CD8+ T-cell response.

Critical role of TLR2 in triggering protective immunity with cyclophilin entrapped in oligomannose-coated liposomes against Neospora caninum infection in mice.

Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii. N. caninum is thought to be a major cause of abortion in cattle worldwide. Given the current situation of drastic economic losses and a lack of efficient control strategies against such parasites, the challenge to develop potent vaccine candidates and technologies remains. We investigated the immune stimulating activity of N. caninum cyclophilin (NcCyp) with and without a formulation with oligomannose-coated-liposomes (OML) as the potential adjuvant. NcCyp-OML activated NF-κB in RAW 264.7 cells and triggered interleukin (IL)-12p40 production from murine peritoneal macrophages. In BALB/c mice, immunization with NcCyp-OML was associated with the production of specific antibodies (IgG1 and IgG2a). The specific antibody (IgG1) against naked NcCyp was also observed after the challenge infection, but it was significantly lower than those of NcCyp-OML. Moreover, significant cellular immune responses were induced, including spleen cell proliferation and interferon-gamma production. The immunization of mice with NcCyp-OML, and to lesser extent with naked NcCyp, induced significant protection against challenge with a lethal dose of N. caninum compared with the PBS control group. This protection was associated with a higher survival rate, slight changes in body weight, and lower clinical score of mice. In addition, the significant protective efficacy of NcCyp-OML was confirmed in another mouse strain, male C57BL/6 mice. The current study revealed the marked contribution of Toll-like receptor 2 (TLR2) to the protective immunity triggered by NcCyp-OML because higher numbers of TLR2-/- mice succumbed to a lethal dose of N. caninum compared with C57BL/6 mice. Moreover, prominent spleen cell proliferation and IFN-γ production was induced in NcCyp-OML-immunized mice by a TLR2-dependent mechanism.

Immunoglobulin G subclass and antibody avidity responses in Malian children immunized with Plasmodium falciparum apical membrane antigen 1 vaccine candidate FMP2.1/AS02A.

BACKGROUND: A malaria vaccine based on Plasmodium falciparum apical membrane antigen 1 (AMA1) elicited strain specific efficacy in Malian children that waned in the second season after vaccination despite sustained AMA1 antibody titers. With the goal of identifying a humoral correlate of vaccine-induced protection, pre- and post-vaccination sera from children vaccinated with the AMA1 vaccine and from a control group that received a rabies vaccine were tested for AMA1-specific immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3, and IgG4) and for antibody avidity. METHODS: Samples from a previously completed Phase 2 AMA1 vaccine trial in children residing in Mali, West Africa were used to determine AMA1-specific IgG subclass antibody titers and avidity by ELISA. Cox proportional hazards models were used to assess correlation between IgG subclass antibody titers and risk of time to first or only clinical malaria episode and risk of multiple episodes. Asexual P. falciparum parasite density measured for each child as area under the curve were used to assess correlation between IgG subclass antibody titers and parasite burden. RESULTS: AMA1 vaccination did not elicit a change in antibody avidity; however, AMA1 vaccinees had a robust IgG subclass response that persisted over the malaria transmission season. AMA1-specific IgG subclass responses were not associated with decreased risk of subsequent clinical malaria. For the AMA1 vaccine group, IgG3 levels at study day 90 correlated with high parasite burden during days 90-240. In the control group, AMA1-specific IgG subclass rise and persistence over the malaria season was modest and correlated with age. In the control group, titers of several IgG subclasses at days 90 and 240 correlated with parasite burden over the first 90 study days, and IgG3 at day 240 correlated with parasite burden during days 90-240. CONCLUSIONS: Neither IgG subclass nor avidity was associated with the modest, strain-specific efficacy elicited by this blood stage malaria vaccine. Although a correlate of protection was not identified, correlations between subclass titers and age, and correlations between IgG subclass titers and parasite burden, defined by area under the curve parasitaemia levels, were observed, which expand knowledge about IgG subclass responses. IgG3, known to have the shortest half-life of the IgG subclasses, might be the most temporally relevant indicator of ongoing malaria exposure when examining antibody responses to AMA1.

Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli.

A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25 nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.