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Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool., Competing Interests: The authors have declared that no competing interests exist.

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4 + and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4 + T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4 + /Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission., Competing Interests: AH is named inventor on patent applications covering malaria vaccines and immunization regimens. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zaric, Marini, Nielsen, Gupta, Mekhaiel, Pham, Elias, Taylor, de Graaf, Payne, Li, Silk, Williams, Hill, Long, Miura and Biswas.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yu, Cao, Gao, Aleem, Liu, Luo, Yan, Xu, Song and Li.)

Background: Transmission-blocking vaccine (TBV) is a promising strategy for malaria elimination. It is hypothesized that mixing or fusing two antigens targeting different stages of sexual development may provide higher transmission-blocking activity than these antigens used individually., Methods: A chimeric protein composed of fragments of Pbg37 and PSOP25 was designed and expressed the recombinant protein in Escherichia coli Rosetta-gami B (DE3). After immunizing mice with individual recombinant proteins Pbg37 and PSOP25, mixed proteins (Pbg37+PSOP25), or the fusion protein (Pbg37-PSOP25), the antibody titers of individual sera were analyzed by ELISA. IFA and Western blot were performed to test the reactivity of the antisera with the native proteins in the parasite. The transmission-blocking activity of the different immunization schemes was assessed using in vitro and in vivo assays., Results: When Pbg37 and PSOP25 were co-administered in a mixture or as a fusion protein, they elicited similar antibody responses in mice as single antigens without causing immunological interference with each other. Antibodies against the mixed or fused antigens recognized the target proteins in the gametocyte, gamete, zygote, and ookinete stages. The mixed proteins or the fusion protein induced antibodies with significantly stronger transmission-reducing activities in vitro and in vivo than individual antigens., Conclusions: There was no immunological interference between Pbg37 and PSOP25. The bivalent vaccines, which expand the portion of the sexual development during which the transmission-blocking antibodies act, produced significantly stronger transmission-reducing activities than single antigens. Altogether, these data provide the theoretical basis for the development of combination TBVs targeting different sexual stages.

Background: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied., Methods: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed., Results: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro., Conclusions: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

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.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PE Δ III-LC3-KDEL3 recombinant protein. In vitro , this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PE Δ III-LC3-KDEL3 reduced the expression of TNF- α , IL-1 β , and NF- κ B in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN- γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN- γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PE Δ III-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Sandra L. Martínez-Hernández et al.)

Avian coccidiosis causes significant economic losses on the global poultry breeding industry. Exploration of new-concept vaccines against coccidiosis has gradually become a research hotspot. In this study, an Enterococcus faecalis strain (MDXEF-1) showing excellent performance isolated from chicken intestinal tract was used as a vector to deliver Eimeria target protein. The plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA harboring dendritic cell-targeting peptide (DCpep) fusion with Eimeria tenella NAΔ3-1E gene (3-1E protein-coding gene without start codon ATG and terminator codon TAA) was electrotransformed into MDXEF-1 to generate the recombinant bacteria MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA in which NAΔ3-1E protein was covalently anchored to the surface of bacteria cells by cell wall anchor (CWA) sequence. The expression of target fusion protein DCpep-NAΔ3-1E-CWA was detected by Western blot. Each chicken was immunized 3 times at 2-wk intervals with live E. faecalis expressing DCpep-NAΔ3-1E fusion protein (DCpep-NAΔ3-1E group), live E. faecalis expressing NAΔ3-1E protein (NAΔ3-1E group), and live E. faecalis containing empty vector only. The 3 immunized groups were then challenged with homologous E. tenella sporulated oocyst after immunizations, and the immune response and protective efficacy in each group were evaluated. The results showed that serum IgG levels, secretory IgA levels in cecal lavage, proportion of CD4+ and CD8α+ cells in peripheral blood, and mRNA expression levels of IL-2 and IFN-γ in the spleen were significantly higher in chickens in the DCpep-NAΔ3-1E group than in chickens of the NAΔ3-1E group (P < 0.05). Oral immunization to chickens with live E. faecalis expressing DCpep-NAΔ3-1E offered more protective efficacy against homologous challenge including significant improved body weight gain, increased oocyst decrease ratio, and reduced average lesion scores in cecum compared with chickens with live E. faecalis expressing NAΔ3-1E protein. These results suggest that recombinant E. faecalis expressing dendritic cell-targeting peptide fusion with E. tenella 3-1E protein could be a potential approach for prevention of Eimeria infection., (Copyright © 2020. Published by Elsevier Inc.)

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (10 8 cells) and HIP (100 μg) for 5 days. Spleens were harvested for flow cytometric and reverse transcriptase PCR analysis. The antileishmanial effect of the combination strategy was associated with induction of a disease-resolving Th1 and Th17 response with simultaneous downregulation of CD4 + CD25 + Foxp3 + (nTreg) cells and CD4 + CD25 - Foxp3 - (Tr1) cells in the spleen. The significant expansion of CD4 + T CM (CD4 + CD44 hi CD11a hi CD62L hi ) cells was a further interesting outcome of this therapeutic strategy in the context of long-term protection of hosts against secondary infection. Toll-like receptor 2 (TLR2) was also found instrumental in this antiparasitic therapy. Induced interleukin-6 (IL-6) production from expanded CD11c + CD8α + (cDC1) and CD11c + CD11b + (cDC2) dendritic cells (DCs) but not from the CD11b + Ly6c + inflammatory monocytes (iMOs), was found critical in the protective expansion of Th17 as evidenced by an in vivo IL-6 neutralization assay. It also promoted the hematopoietic conversion toward DC progenitors (pre-DCs) from common dendritic cell progenitors (CDPs), the immediate precursors, in bone marrow. This novel combinational strategy demonstrated that expansion of Th17 by IL-6 released from CD11c + classical DCs is crucial, together with the conventional Th1 response, to control drug-resistant infection., (Copyright © 2020 American Society for Microbiology.)

The palladacycle complex DPPE 1.2 was previously shown to inhibit Leishmania (Leishmania) amazonensis infection in vitro and in vivo . The present study aimed to evaluate the effect of DPPE 1.2 associated with a recombinant cysteine proteinase, rLdccys1, and the adjuvant Propionibacterium acnes on L. (L.) amazonensis infection in two mouse strains, BALB/c, and C57BL/6. Treatment with this association potentiated the leishmanicidal effect of DPPE 1.2 resulting in a reduction of parasite load in both strains of mice which was higher compared to that found in groups treated with either DPPE 1.2 alone or associated with P. acnes or rLdccys1. The reduction of parasite load in both mice strains was followed by immunomodulation mediated by an increase of memory CD4 + and CD8 + T lymphocytes, IFN-γ levels and reduction of active TGF-β in treated animals. No infection relapse was observed 1 month after the end of treatment in mice which received DPPE 1.2 associated with rLdccys1 or rLdccys1 plus P. acnes in comparison to that exhibited by animals treated with DPPE 1.2 alone. Evaluation of serum levels of AST, ALT, urea, and creatinine showed no alterations among treated groups, indicating that this treatment schedule did not induce hepato or nephrotoxicity. These data indicate the potential use of this association as a therapeutic alternative for cutaneous leishmaniasis caused by L. (L) amazonensis ., (Copyright © 2020 da Silva, Santana, Katz, Garcia, Teixeira, Longo-Maugéri and Barbiéri.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Leishmania rely heavily on glycans to complete their digenetic life cycle in both mammalian and phlebotomine sand fly hosts. Leishmania promastigotes secrete a proteophosphoglycan-rich gel (Promastigote Secretory Gel, PSG) that is regurgitated during transmission and can exacerbate infection in the skin. Here we explored the role of PSG from natural Leishmania-sand fly vector combinations by obtaining PSG from Leishmania (L.) major-infected Phlebotomus (P.) papatasi and P. duboscqi and L. tropica-infected P. arabicus. We found that, in addition to the vector's saliva, the PSG from L. major and L. tropica potently exacerbated cutaneous infection in BALB/c mice, improved the probability of developing a patent cutaneous lesion, parasite growth and the evolution of the lesion. Of note, the presence of PSG in the inoculum more than halved the prepatent period of cutaneous L. tropica infection from an average of 32 weeks to 13 weeks. In addition, L. major and L. tropica PSG extracted from the permissive experimental vector, Lutzomyia (Lu.) longipalpis, also exacerbated infections in mice. These results reinforce and extend the hypothesis that PSG is an important and evolutionarily conserved component of Leishmania infection that can be used to facilitate experimental infection for drug and vaccine screening.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Toxoplasma gondii can infect humans worldwide, causing serious diseases in pregnant women and immunocompromised individuals. T. gondii rhoptry protein 13 (ROP13) is known as one of the key proteins involved in host cell invasion. In this study, we generated virus-like particles (VLPs) vaccine expressing T. gondii rhoptry ROP13 and investigated VLPs vaccine efficacy in mice. Mice immunized with ROP13 VLPs vaccine elicited significantly higher levels of T. gondii-specific IgG, IgG1, IgG2a, and IgA antibody responses following boost immunization and challenge infection, whereas antibody inductions were insignificant upon prime immunization. Differing immunization routes resulted in differing antibody induction, as intranasal immunization (IN) induced greater antibody responses than intramuscular immunization (IM) after boost and challenge infection. IN immunization induced significantly higher levels of IgG and IgA antibody responses from feces, antibody-secreting cells (ASCs), CD4+ T, CD8+ T cells and germinal center B cell responses in the spleen compared to IM immunization. Compared to IM immunization, IN immunization resulted in significantly reduced cyst counts in the brain as well as lesser body weight loss, which contributed to better protection. All of the mice immunized through either route survived, whereas all naïve control mice perished. These results indicate that the ROP13 VLPs vaccine could be a potential vaccine candidate against T. gondii infection.

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.

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01 E vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (r Py CSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4 + and CD8 + T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4 + T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4 + T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates., (Copyright © 2019 American Society for Microbiology.)

Purpose: Leishmaniasis, as one of the most important vector-borne and zoonotic diseases, can be seen in different forms and is more prevalent in developing countries worldwide. Due to the absence of effective strategies in its prevention, treatment, and control, investigation of effective control strategies against the disease is necessary. In this research, we evaluated the immunogenicity of a cold-adapted laboratory strain of Leishmania major (LMC) in the mouse model., Methods: Twenty BALB/c mice were divided into two groups. LMC group received 4 × 10 6 of LMC strain in 0.5 ml DMEM, and VLM group, as the control group, received 0.5 ml Dulbecco's modified Eagle's medium. Both groups were challenged with virulent L. major 3 weeks after inoculation., Results: The data obtained from the analysis of immune responses and histopathological changes interestingly revealed protection against L. major in immunized mice. Compared with the VLM group, the mice immunized with LMC strain of L. major in the LMC group showed a significant increase in IFN-γ and IgG2a levels (P < 0.05) which are important indexes for Th1-related immune responses. Additionally, significant differences in concentration of IgG1 and IgG total before and after the challenge was observed in LMC group (P < 0.05). Furthermore, the immunized mice showed a significant reduction in mean sizes of skin lesion and liver damage compared to the VLM group., Conclusion: Based on the present findings on immunogenicity of LMC strain, it seems this strain is able to induce both humoral and cellular immunity and a significant protection against L. major in the mouse model.

Background: Toxoplasma gondii has a comprehensive impact on a great range of warm-blood mammals, in which one-third of the population all over the world is involved. Dense granular proteins, regarded as GRA family, mediating substantial interface between host cell cytoplasm and parasite, are widely studied for preventing the infection of T. gondii., Purpose: As is handled in our study, the effect of intramuscularly injecting the genetic vaccine pEGFP-C1/GRA41 encoding a novel dense granule protein, GRA41, was evaluated., Methods: At the beginning, bioinformatics analysis was used to evaluate epitopes of both B cells and T cells on the GRA41 protein of T. gondii. Afterwards, recombinant plasmids (pEGFP-C1/GRA41) were injected into BALB/c mice and the quantity of IgG and its subclass IgG2a remarkably increased. IFN-γ, distinctive from the other cytokines (IL-4, and IL-10), was significant in growth. Afterwards, the intraperitoneal challenge was executed for recording survival time with tachyzoites with high virulence (in RH strain) and counting the number of brain cysts was carried out after the infection of PRU strain (low virulence)., Results: In pEGFP-C1/GRA41 group, the survival period was significantly longer (13.3 ± 3.37 days) after tachyzoites attack with the RH strain in high virulence, compared with the other groups (less than 8 days). Additionally, the cyst quantity is remarkably lower and the rate of reduction could reach 59.34%., Conclusion: All the results indicated effective protection of DNA vaccine encoding GRA41 against T. gondii.

Toxoplasma gondii, an intracellular parasitic protozoan, is capable of infecting man and all warm-blooded animals. Cell-mediated immunity is vital in mounting protective responses against T. gondii infection. Recent studies have shown that T-helper (Th) 17 responses may play a key role in parasite control. In this current study, we constructed a DNA vaccine encoding T. gondii ROP13 in a pcDNA vector. Groups of BALB/c mice were immunized intramuscularly with pcROP13 or controls and challenged with the RH strain of T. gondii. The results showed that immunization with pcROP13 could elicit an antibody response against T. gondii. The expression of the canonical Th17 cytokines, interleukin (IL)-17 and IL-22, were significantly increased after immunization with pcROP13 compared with control groups ( p < 0.05). Furthermore, vaccination resulted in a significant decrease in parasite load ( p < 0.05). The induction of Th17 related cytokines, using a ROP13 DNA vaccine, against T. gondii should be considered as a potential vaccine approach for the control of toxoplasmosis., (© 2018 Wiley Periodicals, Inc.)

The current therapy for the treatment of leishmaniasis is unsatisfactory because it has multiple side effects, and resistance has been reported among the parasites that cause these diseases. Numerous efforts have been made to develop new candidates for vaccines. In recent years, particles of biodegradable polymers have been proposed as vehicles to transport and protect antigens, proteins, drugs and vaccines. In this work, the oil/water (o/w) single emulsion-solvent evaporation technique was used to prepare PLGA biodegradable particles. The encapsulation of two hypothetical proteins from Leishmania panamensis was performed to validate the proposed method. For this validation, different concentrations (50, 100, 150, 200, 250, 500, and 750 μg/ml) of both proteins were encapsulated into PLGA particles, and the particle sizes and shapes were evaluated by optical microscopy and scanning electron microscopy (SEM), respectively. The release of proteins was confirmed by SDS-PAGE and Western blot analyses. The integrity of both proteins was conserved, and they were released from day one until day 15, with a maximum amount of 46 ± 4.25% for the LpanUA.27.1260 protein and 26.19 ± 3.41% for LpanUA.22.1860. Additionally, the protective efficacy of one of these encapsulated proteins was evaluated in vivo using BALB/c mice infected with L. panamensis. Therefore, the encapsulation of proteins is presented here as an excellent alternative to evaluate the antigenicity of proteins from parasites of medical importance such as L. panamensis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antibodies to Circumsporozoite protein (CSP) confer protection against controlled human malaria infection (CHMI) caused by the parasite Plasmodium falciparum. Although CSP is highly immunogenic, it does not induce long lasting protection and efforts to improve CSP-specific immunological memory and duration of protection are underway. We have previously reported that the clinical grade CSP vaccine FMP013 was immunogenic and protective against malaria challenge in mice when combined with the Army Liposomal Formulation adjuvant containing immune modulators 3D-PHAD™ and QS21 (ALFQ). To move forward with clinical evaluation, we now report the safety, toxicity and immunogenicity of clinical grade FMP013 and ALFQ in Rhesus macaques. Three groups of Rhesus (n = 6) received half or full human dose of FMP013 + ALFQ on a 0-1-2 month schedule, which showed mild local site reactions with no hematologic derangements in red blood cell homeostasis, liver function or kidney function. Immunization induced a transient systemic inflammatory response, including elevated white blood cell counts, mild fever, and a few incidences of elevated creatine kinase, receding to normal range by day 7 post vaccination. Optimal immunogenicity in Rhesus was observed using a 1 mL ALFQ + 20 µg FMP013 dose. Doubling the FMP013 antigen dose to 40 µg had no effect while halving the ALFQ adjuvant dose to 0.5 mL lowered immunogenicity. Similar to data generated in mice, FMP013 + ALFQ induced serum antibodies that reacted to all regions of the CSP molecule and a Th1-biased cytokine response in Rhesus. Rhesus antibody response to FMP013 + ALFQ was found to be non-inferior to historical benchmarks including that of RTS,S + AS01 in humans. A four-dose GLP toxicity study in rabbits confirmed no local site reactions and transient systemic inflammation associated with ALFQ adjuvant administration. These safety and immunogenicity data support the clinical progression and testing of FMP013 + ALFQ in a CHMI trial in the near future., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, and Quillaja saponaria Molina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity of Escherichia coli -expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in female Anopheles stephensi mosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development in A. stephensi However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) in A. stephensi It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria., (Copyright © 2019 American Society for Microbiology.)

Background: In holoendemic areas, children suffer the most from Plasmodium falciparum malaria, yet newborns and young infants express a relative resistance to both infection and severe malarial disease (SM). This relative resistance has been ascribed to maternally-derived anti-parasite immunoglobulin G; however, the targets of these protective antibodies remain elusive., Methods: We enrolled 647 newborns at birth from a malaria-holoendemic region of Tanzania. We collected cord blood, measured antibodies to Plasmodium falciparum Schizont Egress Antigen-1 (PfSEA-1), and related these antibodies to the risk of severe malaria in the first year of life. In addition, we vaccinated female mice with PbSEA-1, mated them, and challenged their pups with P. berghei ANKA parasites to assess the impact of maternal PbSEA-1 vaccination on newborns' resistance to malaria., Results: Children with high cord-blood anti-PfSEA-1 antibody levels had 51.4% fewer cases of SM compared to individuals with lower anti-PfSEA-1 levels over 12 months of follow-up (P = .03). In 3 trials, pups born to PbSEA-1-vaccinated dams had significantly lower parasitemia and longer survival following a P. berghei challenge compared to pups born to control dams., Conclusions: We demonstrate that maternally-derived, cord-blood anti-PfSEA-1 antibodies predict decreased risk of SM in infants and vaccination of mice with PbSEA-1 prior to pregnancy protects their offspring from lethal P. berghei challenge. These results identify, for the first time, a parasite-specific target of maternal antibodies that protect infants from SM and suggest that vaccination of pregnant women with PfSEA-1 may afford a survival advantage to their offspring., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The pathogenesis of cerebral malaria is biologically complex and involves multi-factorial mechanisms such as microvascular congestion, immunopathology by the pro-inflammatory cytokine and endothelial dysfunction. Recent data have suggested that a pleiotropic T-cell immunomodulatory protein (TIP) could effectively mediate inflammatory cytokines of mammalian immune response against acute graft-versus-host disease in animal models. In this study, we identified a conserved homologue of TIP in Plasmodium berghei (PbTIP) as a membrane protein in Plasmodium asexual stage. Compared with PBS control group, the pathology of experimental cerebral malaria (ECM) in rPbTIP intravenous injection (i.v.) group was alleviated by the downregulation of pro-inflammatory responses, and rPbTIP i.v. group elicited an expansion of regulatory T-cell response. Therefore, rPbTIP i.v. group displayed less severe brain pathology and feverish mice in rPbTIP i.v. group died from ECM. This study suggested that PbTIP may be a novel promising target to alleviate the severity of ECM.

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.

Leishmaniasis is an expanding health threat worldwide complicated by the absence of an effective vaccine. We investigated transcutaneous immunization (TCI) as a needle-free immunization route which exploits the abundance of antigen presenting cells in the skin to induce both mucosal and systemic immunity. Leishmania (L.) major soluble antigens (SLA) or recombinant Leishmania homolog of receptors for activated C-kinase (rLACK) antigens were delivered transcutaneously together with cholera toxin (CT), to BALB/c mice. Mice were immunized at weeks 1, 4, and 7 with PBS, CT, SLA/CT or rLACK/CT. Two weeks after the final boost, antigen-specific IgG titers, IFN-γ ELISpot, and cytokine levels were assessed in half of the mice and the remainder were challenged with an intradermal (ear) injection of 5 × 10 4 L. major metacyclic parasites. Mice were monitored weekly and sacrificed after 7 weeks to assess the parasite burden and to study the ear lesion immunohistopathology. Our results show that TCI with SLA or rLACK yielded high levels of anti-SLA, anti-rLACK and anti-CT IgG antibodies. A Th1-type of immune response was demonstrated with a high frequency of IFN-γ secreting cells, high levels of IFN-γ production, and lower levels of IL-10 resulting in a high IFN-γ/IL-10 ratio in mice immunized with SLA/CT or rLACK/CT. After parasite challenge, rLACK immunization was not associated with protection. In addition, SLA/CT immunized mice had larger ear lesions and an increased parasite load in the ear. Immunohistochemistry of ear biopsies stained for nitric oxide synthase revealed that staining intensity was diminished in the SLA/CT group compared to the control group. This finding suggested that less parasite killing occurred at the site of the infection. In conclusion, despite a strong Th1 type profile induced by TCI, exacerbation of infection occurred after challenge with L. major. This also correlated with low induction of nitric oxide., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

OprI is an outer membrane lipoprotein from Pseudomonas aeruginosa, and when fused to a recombinant antigen, will exert adjuvant properties by engaging Toll-like receptor 2, leading to dendritic cell activation. Previous studies have shown that the Neospora caninum (Nc) antigens NcPDI, NcROP2 and NcROP40 are implicated in host cell interactions and are promising vaccine candidates. In two independent experiments, the efficacy of a polyvalent vaccine formulation composed of OprI-NcPDI, OprI-NcROP2 and OprI-NcROP40 (collectively named O-Ags) was assessed in non-pregnant and pregnant Balb/c mouse models challenged with tachyzoites of the high-virulence isolate Nc-Spain7. Parameters that were investigated were clinical signs, fertility, parasite burden in adult mice, humoral and cellular immune responses at different time-points prior to and after challenge infection, vertical transmission and post-natal survival of offspring mice, all to explore potential correlations with efficacy. Vaccination of mice with O-Ags induced a mixed Th1/Th2 immune response in adult mice and led to significantly increased protection against cerebral infection. Vaccination with O-Ags also resulted in reduced vertical transmission, and postnatal disease in offspring was significantly inhibited at a rate not observed in mice infected with a high-virulence isolate to date. However, O-Ags mixed with TLR ligands targeting TLR3 and TLR7, which are known to induce clear Th1-biased responses, or vaccination with OprI fused to the non-N. caninum antigen ovalbumin (OprI-OVA) did not confer protection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The international spread of infectious diseases is a global problem of health security. Vaccination is one of the most successful and profitable health interventions. Oral immunization has significant advantages over the widely used parental vaccines. Intestinal and free-living protozoa express on their surface a dense layer of proteins that protect them from hostile environmental conditions. The use of variable surface proteins (VSPs), such as those of the intestinal protozoan Giardia lamblia, is a feasible mechanism for the generation of oral vaccines, since they are highly immunogenic as well as resistant to changes in pH and proteases. In a recently published article, we showed that these properties of VSPs can be exploited to protect and enhance the immunogenicity of vaccine antigens, thus enabling their oral administration. We recently generated an oral vaccine against influenza virus composed of virus-like particles (VLPs) containing VSPs of G. lamblia and the HA antigen (viral hemagglutinin) in its envelope. When administered orally to mice, these coated particles elicit HA-specific humoral (systemic and local) and cellular responses, without the need of any additional adjuvant. Treated mice are protected against viral challenge as well as against the development of tumors expressing the HA vaccine antigen.

Pfs25 is a malaria transmission-blocking vaccine antigen candidate, but its apparently limited immunogenicity in humans has hindered clinical development. Here, we show that recombinant, polyhistidine-tagged (his-tagged) Pfs25 can be mixed at the time of immunization with pre-formed liposomes containing cobalt porphyrin-phospholipid, resulting in spontaneous nanoliposome antigen particleization (SNAP). Antigens are stably presented in uniformly orientated display via his-tag insertion in the cobalt porphyrin-phospholipid bilayer, without covalent modification or disruption of antigen conformation. SNAP immunization of mice and rabbits is well tolerated with minimal local reactogenicity, and results in orders-of-magnitude higher functional antibody generation compared with other 'mix-and-inject' adjuvants. Serum-stable antigen binding during transit to draining lymph nodes leads to enhanced antigen uptake by phagocytic antigen-presenting cells, with subsequent generation of long-lived, antigen-specific plasma cells. Seamless multiplexing with four additional his-tagged Plasmodium falciparum polypeptides induces strong and balanced antibody production, illustrating the simplicity of developing multistage particulate vaccines with SNAP immunization.

Background: Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis., Results: The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas., Conclusions: The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

Leishmania elongation factor 2 (EF-2) has been previously identified as a T H 1-stimulatory protein. In this study, we assayed the protective potential of the N-terminal domain of EF-2 (N-LiEF-2, 1-357 aa) that has been predicted to contain several overlapping MHC class I and II-restricted epitopes injected in the form of dendritic cell (DC)-based vaccine. Ex vivo pulsing of DCs with the recombinant N-LiEF-2 domain along with CpG oligodeoxynucleotides (ODNs) resulted in their functional differentiation. BALB/c vaccinated with CpG-triggered DCs pulsed with N-LiEF-2 were found to be the most immune-reactive in terms of induction of DTH responses, increased T cell proliferation and IL-2 production. Moreover, vaccination induced antigen-specific T H 1 type immune response as evidenced by increased IFN-γ and TNFα levels followed by a significant increase of nitrite (NO) and reactive oxygen species (ROS) in splenocyte cultures. Vaccinated mice showed a pronounced decrease in parasite load in spleen and liver when challenged with L. infantum, increased expression of Stat1 and Tbx21 mRNA transcripts versus reduced expression of Foxp3 transcripts and were able to produce significantly elevated levels of IL-2, IFN-γ and TNFα but not IL-10 compared to non-vaccinated mice. Both antigen and parasite-specific CD4 + T and CD8 + T cells contributed to the IFN-γ production indicating that both subtypes contribute to the resistance to infection and correlated with robust nitrite generation, critical in controlling Leishmania infection. Together, these findings demonstrated the immunogenic as well as protective potential of the N-terminal domain of Leishmania EF-2 when given with CpG-triggered DCs representing a basis for the development of rationalized vaccine against leishmaniasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Plasmodium falciparum malaria pathogenesis is tied to the sequestration of parasites in the microvasculature. Parasite sequestration leading to severe malaria is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) binding to endothelial protein C receptor (EPCR) via its CIDRα1 domains. CIDRα1 domains are targets of naturally acquired immunity, and a vaccine eliciting antibodies inhibiting the EPCR binding of CIDRα1 could potentially prevent disease and death from malaria. CIDRα1 domains have diversified in sequence to escape immune recognition but preserved structure to maintain EPCR binding. The EPCR-binding CIDRα1 domains separate into six major sequence types predicted to form a conserved structure in which only the amino acids essential for EPCR binding are highly conserved. Here, we investigated whether antibodies elicited by vaccination with single or multiple recombinant CIDRα1 domains are able to bind and inhibit diverse CIDRα1 domains. We found that EPCR binding-inhibitory antibodies to CIDRα1 variants closely related to those used for vaccination are readily elicited, whereas antibodies binding distant CIDRα1 variants are sporadically generated and are rarely inhibitory. Despite this, sequence similarity correlated poorly with the ability of induced antibodies to inhibit across diverse variants, and no continuous sequence regions of importance for cross-inhibitory antibodies could be identified. This suggested that epitopes of cross-variant inhibitory antibodies were predominantly conformational. Vaccination with immunogens engineered to focus immune responses to specific epitopes or an optimal choice of multiple CIDRα1 variants may improve elicitation of broadly reactive and inhibitory antibody responses., (Copyright © 2018 American Society for Microbiology.)

Toxoplasma gondii rhoptry proteins (TgROPs) have been considered the main targets and indicator molecules for immune diagnosis and prophylaxis because they present during the initial process of invasion. The aim of this study was to evaluate the protective effect of a TgROP35 DNA vaccine on experimental mice subjected to T. gondii challenge. The effect of intramuscularly injecting the genetic vaccine pVAX-ROP35 into BALB/c mice was evaluated, by first inserting the TgROP35 sequence into the pVAX I eukaryotic expression vector. The levels of IgG, IgG1 and IgG2a in pVAX-ROP35-vaccinated animals were integrally increased. Cytokine profile analyses revealed that IFN-γ, IL-2 and IL-10 levels were significantly increased, while there were no significant differences in IL-4 expression between the pVAX-ROP35 immunized and control groups. Additionally, we found that immunization with pVAX-ROP35 significantly prolonged the survival time (13.90 ± 1.97 days) of mice after challenge infection with the virulent T. gondii RH strain compared with that in non-vaccinated control animals (mice died within 10 days). Moreover, the number of brain cysts (1450 ± 287) in the animals subjected to pVAX-TgROP35 vaccination decreased remarkably (P <  0.05) compared to that in the blank control mice (2333 ± 473). Furthermore, the size of brain cysts in the pVAX-TgROP35 group was significantly smaller than that in the blank control, PBS and pVAXI groups. The findings indicated that intense cell-mediated and humoural immunity was triggered and that defence mechanisms against T. gondii were partially induced after vaccination by TgROP35., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vivax malaria remains one of the most serious and neglected tropical diseases, with 132 to 391 million clinical cases per year and 2.5 billion people at risk of infection. A vaccine against Plasmodium vivax could have more impact than any other intervention, and the use of a vaccine targeting multiple antigens may result in higher efficacy against sporozoite infection than targeting a single antigen. Here, two leading P. vivax preerythrocytic vaccine candidate antigens, the P. vivax circumsporozoite protein (PvCSP) and the thrombospondin-related adhesion protein (PvTRAP) were delivered as a combined vaccine. This strategy provided a dose-sparing effect, with 100% sterile protection in mice using doses that individually conferred low or no protection, as with the unadjuvanted antigens PvTRAP (0%) and PvCSP (50%), and reached protection similar to that of adjuvanted components. Efficacy against malaria infection was assessed using a new mouse challenge model consisting of a double-transgenic Plasmodium berghei parasite simultaneously expressing PvCSP and PvTRAP used in mice immunized with the virus-like particle (VLP) Rv21 previously reported to induce high efficacy in mice using Matrix-M adjuvant, while PvTRAP was concomitantly administered in chimpanzee adenovirus and modified vaccinia virus Ankara (MVA) vectors (viral-vectored TRAP, or vvTRAP) to support effective induction of T cells. We examined immunity elicited by these vaccines in the context of two adjuvants approved for human use (AddaVax and Matrix-M). Matrix-M supported the highest anti-PvCSP antibody titers when combined with Rv21, and, interestingly, mixing PvCSP Rv21 and PvTRAP viral vectors enhanced immunity to malaria over levels provided by single vaccines., (Copyright © 2018 Atcheson et al.)

E. mitis is ubiquitous in clinical coccidiosis caused by mixed infection of Eimeria species and the infection by E. mitis usually significantly impairs productivity of the infected chickens. To date, however, few protective antigens from E. mitis have been reported. In this study, the molecular characterization and protective efficacy of microneme 3 of Eimeria mitis (EmiMIC3) were analyzed. EmiMIC3 gene was cloned from sporozoites of E. mitis and its MARs (microneme adhesive repeats domain) were predicted. Recombinant EmiMIC3 (rEmiMIC3) was expressed in E. coli and purified and then was analyzed by western blot with anti-E. mitis chicken serum. Meanwhile, native EmiMIC3 from sporozoites was analyzed by anti-rEmiMIC3 rat serum. The expressions of EmiMIC3 in E. mitis sporozoites and merozoites were analyzed by immunofluorescence assay. The rEmiMIC3-induced changes of T lymphocytes subpopulation, serum cytokines and IgY levels and the protective efficacy of rEmiMIC3 were determined in animal experiments. The results showed that the deduced open reading frame (ORF) of EmiMIC3 was composed of 1145 amino acids, possessing 9 MARs. EmiMIC3 gene was submitted to GenBank (accession number: MG888670). EmiMIC3 could express in sporozoites and merozoites respectively and located at the apex of E. mitis sporozoite. Western blot assay revealed that the rEmiMIC3 could be recognized by serum of chicken infected by E. mitis and the native EmiMIC3 from sporozoites could also be recognized by rat serum against rEmiMIC3. Following vaccination with rEmiMIC3, higher levels of IL-10, IFN-γ, TGF-βand IL-17, higher proportions of CD4+/CD3+ and CD8+/CD3 + T lymphocytes and higher level of IgY antibody were induced compared to the controls. Vaccination with rEmiMIC3 prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection. Our result not only enriches protective candidate antigen of E. mitis, but also provides available protective antigen of E. mitis for the development of multivalent vaccines against infection caused by mixture of Eimeria species in clinical coccidiosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the present study, we have investigated the protective effect of a heterologous prime-boost strategy with priming plasmid DNA followed by recombinant adenovirus, both expressing BmAMA1, against Babesia microti infection. Four groups consisting of 3 hamsters per group were immunized with pBmAMA1/Ad5BmAMA1, pNull/Ad5BmAMA1, pBmAMA1/Ad5Null and pNull/Ad5Null, followed by challenge infection with B. microti. Our results showed that hamsters immunized with plasmid and adenovirus expressing BmAMA1 developed a robust IgG and IgG2a antibody response against BmAMA1, suggesting the DNA vaccine or viral vector vaccine tend to induce a Th1-biased response. Compared to the control hamsters, the hamsters vaccinated either with the prime-boost strategy or one of the two "vaccines" exhibited no significant protection against B. microti challenge. Although a slight difference in terms of parasitemia and hematocrit values at days 14-16 post challenge infection was observed, no other statistical difference was detected. Our results indicate that the prime-boost vaccination strategy of injection of plasmid and adenovirus expressing BmAMA1 is not efficient in protecting against B. microti infection.

Influenza viruses are globally important respiratory pathogens with high degree of morbidity and mortality during annual epidemics. Influenza A vaccination has been challenged due to genetic evolution. M2 protein is the surface antigen of the virus and facilitates virus entry into the host cells. N-terminal 24 amino acid residues that constitute the extracellular domain of M2 protein (M2e) show remarkable conservation among all subtypes of influenza A viruses. The aim of the present study was to investigate the effects of using HSP70 as an adjuvant fused to three tandem repeats of M2e (3M2e) to enhance the immune responses against influenza A challenge. The mice were immunized three times by intradermal inoculations of 3M2e alone or in combination with Alum adjuvant or in fused form to HSP70. The specific immune responses were evaluated by measuring the serum antibody titers, lymphocyte proliferation, as well as Th1 and Th2 cytokines. The results showed that, although 3M2e with no adjuvant could induce secretion of specific antibodies, significantly higher humoral immune responses were stimulated in combination with Alum adjuvant (p < 0.05). Moreover, analysis of specific immune responses revealed that the 3M2e-HSP70 chimer protein mainly stimulated IgG2a and IFN-γ responses indicating aTh1 bias which shows the ability of HSP as a powerful adjuvant for activation of cellular immune responses. This was supported by a higher IgG2a/IgG1 ratio, significantly increased IL-4 production and lymphocyte proliferation (P ˂ 0.001) compared with mice vaccinated with 3M2e alone or supplemented with Alum, suggesting a mixture of Th1 and Th2 type cellular immune response with a Th1 bias. The findings of this study indicated that 3M2e-HSP70 enhances humoral and cellular immune responses and improves immune protection against influenza challenge in mice. Thus, it has the potential to be used as a promising vaccine candidate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, a Leishmania hypothetical protein, LiHyS, was evaluated regarding its antigenicity, immunogenicity and protective efficacy against visceral leishmaniasis (VL). Regarding antigenicity, immunoblottings and an enzyme-linked immunosorbent assay using human and canine sera showed high sensitivity and specificity values for the recombinant protein (rLiHyS) in the diagnosis of VL. When evaluating the immunogenicity of LiHyS, which is possibly located in the parasite's flagellar pocket, proliferative assays using peripheral blood mononuclear cells from healthy subjects or VL patients showed a high proliferative index in both individuals, when compared to the results obtained using rA2 or unstimulated cultures. Later, rLiHyS/saponin was inoculated in BALB/c mice, which were then challenged with Leishmania infantum promastigotes. The vaccine induced an interferon-γ, interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor production, which was maintained after infection and which was associated with high nitrite and IgG2a antibody levels, as well as low IL-4 and IL-10 production. Significant reductions in the parasite load in liver, spleen, bone marrow and draining lymph nodes were found in these animals. In this context, the present study shows that the rLiHyS has the capacity to be evaluated as a diagnostic marker or vaccine candidate against VL.

Malaria is a life-threatening infectious disease and continues to be a major public health crisis in many parts of the tropical world. Plasmodium falciparum is responsible for the majority of mortality and morbidity associated with malaria. During the intraerythrocytic cycle, P. falciparum releases three proteins with high histidine content as follows: histidine-rich protein 1 (HRP1), histidine-rich protein 2 (HRP2), and histidine-rich protein 3 (HRP3). Currently, most of the diagnostic tests of P. falciparum infection target HRP2, and a number of monoclonal antibodies (mAbs) against HRP2 have been developed for use in HRP2 detection and quantification. When parasites have HRP2 deletions, the detection of HRP3 could augment the sensitivity of the detection system. The combination of both HRP2 and HRP3 mAbs in the detection system will enhance the test sensitivity. In the HRP quantitative enzyme-linked immunosorbent assay (ELISA), both HRP2 and HRP3 contribute to the result, but the relative contribution of HRP2 and HRP3 was unable to investigate, because of the nonavailability of HRP3 specific antibody ELISA. Hence an ELISA test system based on HRP3 is also essential for detection and quantification. There is not much documented in the literature on HRP3 antigen and HRP3 specific mAbs and polyclonal antibodies (pAbs). In the present study, recombinant HRP3 was expressed in Escherichia coli and purified with Ni-NTA agarose column. The purified rHRP3 was used for the generation and characterization of monoclonal and pAbs. The purification of monoclonal and pAbs was done using a mixed-mode chromatography sorbent, phenylpropylamine HyperCel™. With the purified antibodies, a sandwich ELISA was developed. The sandwich ELISA method was explored to detect and quantify HRP3 of P. falciparum in the spent medium. The generated mAbs could be potentially used for the detection and quantification of P. falciparum HRP3.

Despite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of soluble Leishmania donovani antigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy against L. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treated Leishmania-infected hamsters and peripheral blood mononuclear cells of Leishmania patients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treated Leishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses in Leishmania patients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

About one-third of the world population is prone to have infection with T. gondii, which can cause toxoplasmosis in the developing fetus and in people whose immune system is compromised through disease or chemotherapy. Surface antigen-1 (SAG1) is the candidate of vaccine against toxoplasmosis. Recent advances in biotechnology and nano-pharmaceuticals have made possible to formulate nanospheres of recombinant protein, which are suitable for sub-unit vaccine delivery. In current study, the local strain was obtained from cat feces as toxoplasma oocysts. Amplified 957 bp of SAG1 was cloned into pGEM-T and further sub-cloned into pET28-SAG1. BL21 bacteria were induced at different concentrations of isopropyl β-d-1-thiogalactopyranoside for the expression of rSAG1 protein. An immunoblot was developed for the confirmation of recombinant protein expression at 35 kDa that was actually recognized by anti-HIS antibodies and sera were collected from infected mice. PLGA encapsulated nanospheres of recombinant SAG1 were characterized through scanning electron microscopy. Experimental mice were intraperitoneally immunized with rSAG1 protein and intra-nasally immunized with nanosphere. The immune response was evaluated by indirect ELISA. In results intra-nasally administered rSAG1 in nanospheres appeared to elicit elevated responses of specific IgA and IgG2a than in control. Nanospheres of rSAG1 are found to be a bio-compatible candidate for the development of vaccine against T. gondii.

Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens., (© M. Yan et al., published by EDP Sciences, 2018.)

This study is aimed to evaluate the protective effect of L. donovani intracellular serine protease (SP-Ld) in combination with Freund's adjuvant and liposomal formulations against experimental visceral leishmaniasis (VL). The animals were immunized with SP-Ld in combination with adjuvant and evaluated for its immunogenicity and protective efficacy against Leishmania donovani. The infection was initially assessed by microscopic examination. Immunogenicity of SP-Ld was measured by detecting protease specific-IgG, IgG1 and IgG2a levels by ELISA. Cytokines levels were measured by ELISA and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The vaccine efficacy of SP-Ld was also evaluated by measuring antibody response and survival potency in hamster model. SP-Ld vaccinated Balb/c mice resulted significant reduction of parasite burden with increased levels of IgG2a and decreased levels of IgG1. SP-Ld vaccination also induced Th1 type immune response with the rise of IL-12, IFN-γ and TNF-α with decreased levels of IL-10 and TGF-β. Importantly, liposomal incorporated SP-Ld exerted better protection rather than in combination with Freund's adjuvant. Additionally, liposome encapsulated SP-Ld vaccinated hamsters continued to survive beyond 8 months against virulent L. donovani post challenge. Overall, these findings demonstrated SP-Ld as an effective immunogen which opens a new perspective for the generation of potential vaccine candidate against leishmaniasis., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Infections with the protozoan parasite Toxoplasma gondii, which are common around the world, can lead to congenital infections in humans. T. gondii surface antigen protein 5B (SAG5B) and SAG5C are potential stimulators of humoral and cellular immune responses. In this study, a multi-antigenic DNA vaccine constructed to express T. gondii SAG5B and SAG5C proteins simultaneously was used to immunize BALB/c mice to evaluate the protective efficacy of the vaccine. IgG antibody and gamma interferon (IFN-γ) cytokine production in the pSAG5B/SAG5C DNA vaccine group were significantly higher (0.853±0.103 and 915.2±106.9, respectively) than in the single DNA vaccine groups (pSAG5B, 0.667±0.109 and 598.3±74.9, respectively; pSAG5C, 0.696±0.092 and 623.7±95.5, respectively). After a lethal challenge with 1×10 4 RH strain tachyzoites, the survival time of the mice (17days) immunized with pSAG5B/SAG5C was longer than that of the single-gene-immunized mice (12days) or the control mice (6days). Moreover, after intragastric infection with 20 T. gondii PRU (low virulence) strain cysts, the number of brain cysts in the pSAG5B/SAG5C-vaccinated mice was only 25% of the number for the PBS-injected mice. Our findings indicate that, in comparison with the other mouse groups, the multi-antigenic DNA vaccine (pSAG5B/SAG5C) significantly induced immune responses and improved the protection against challenge with T. gondii in the host animals., (Copyright © 2017. Published by Elsevier B.V.)

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens., (Copyright © 2017 Elsevier B.V. All rights reserved.)