Vaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii

doi:10.1016/j.vaccine.2014.01.095

Vaccine

Volume 32, Issue 16, 1 April 2014, Pages 1781-1785

https://doi.org/10.1016/j.vaccine.2014.01.095 Get rights and content

Highlights

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We confirmed the immunogenicity of TgPF in mice after Toxoplasma gondii infection.
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TgPF-OML administration increased the survival of mice after Toxoplasma gondii infection.
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TgPF-OML administration reduced the parasite burden in mouse brains after infection.
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Immunization with TgPF-OML induced TgPF-specific interferon-γ and IgG production.

Abstract

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals and birds, causing toxoplasmosis. Several types of vaccines against T. gondii have been developed, but these have limitations in terms of their safety and inadequate efficacy. T. gondii profilin (TgPF) is a potential immunodominant antigen for a candidate vaccine. In this study, we encapsulated TgPF in oligomannose-coated liposomes (OMLs) to evaluate the immune response induced by this vaccine. C57BL/6 mice were immunized with TgPF-OML three times at 14-day intervals and challenged with T. gondii. TgPF-OML increased the survival of the mice and reduced the parasite burden in their brains after T. gondii infection. Immunization with TgPF-OML also induced TgPF-specific interferon-γ production and IgG antibodies in mice. Our results demonstrate that OML-encapsulated TgPF triggers strong humoral and cellular responses against T. gondii, and that TgPF-OML is a candidate vaccine that warrants further development.

Introduction

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds, causing toxoplasmosis [1]. An effective vaccine against T. gondii is an ideal strategy for controlling acute or chronic toxoplasmosis and prevent its consequences because no drugs are available that effectively eliminate the parasite from the infected host. In previous studies, we showed that liposomes coated with a neoglycolipid constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3–DPPE) induced a strong T-helper 1 (Th1) immune response against the encapsulated antigen following the administration of Man3–DPPE-coated liposomes (oligomannose-coated liposomes, OMLs) into the peritoneal cavities of mice [2]. The action of OMLs as an effective adjuvant has been confirmed with Leishmania major [2] and Neospora caninum infections [3], [4], [5].

Toxoplasma gondii profilin (TgPF) is a potent agonist of the innate immune system through its recognition by Toll-like receptor 11 (TLR11). This receptor plays a critical role in the induction of the host protective cytokine interleukin 12 (IL-12) [6]. TgPF has been shown to be an immunodominant antigen in the CD4⁺ T-cell response to the pathogen [7]. However, no study has evaluated the potential use of TgPF as a vaccine against T. gondii.

In this study, we examined the immunogenicity and protective efficacy of TgPF encapsulated in OMLs and evaluated the potential use of TgPF as a vaccine in C57BL/6 mice challenged with a lethal infective dose of T. gondii strain PLK.

Section snippets

Mice and parasites

Seven-week-old female C57BL/6 mice were purchased from Clea Japan (Tokyo, Japan). All the mice were treated under the guiding principles for the care and use of research animals promulgated by Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. T. gondii strain PLK (type II strain) was cultured and purified as previously described [8].

Preparation of recombinant protein and liposomes

The TgPF gene was amplified from cDNA by PCR using oligonucleotide primers that included an EcoRI site in the forward primer 5′-ATG AAT TCA

Immunogenicity of TgPF in Toxoplasma gondii-infected mice

Significantly elevated levels of IFN-γ were detected in the spleen cells from T. gondii-infected mice stimulated with 50 μg/ml TLA (100.0 ± 18.95 ng/ml) or 50 μg/ml TgPF (2.8 ± 0.75 ng/ml), but not in those stimulated with 50 μg/ml TgCyp18 (0.2 ± 1.17 ng/ml) (Fig. 1A and B).

Survival rates of immunized mice after challenge with Toxoplasma gondii

The survival rate of the mice immunized with TgPF-OML (66.7%) was significantly higher than that of mice treated with PBS (25.0%), OML (25.0%), or TgPF (16.7%) (Fig. 1C). The parasite burden in the brains of the surviving mice in the

Discussion

In this study, we have demonstrated that subcutaneous immunization with TgPF-OML induces a protective response against T. gondii infection in mice and that this is caused by the strong induction of a TgPF-specific Th1 immune response, the key event in the control of toxoplasmosis [11]. Administration of TgPF-OML also induced the production of antigen-specific IgG antibodies, especially IgG2c. In C57BL/6 mice, antibodies of the IgG2c subclass are the predominant isotype involved in

Conflicts of interest statement

The authors have no potential conflicts of interest to report.

Acknowledgments

We thank Youko Matsushita, Megumi Noda, and Yoshie Imura (National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine) for their excellent technical assistance. This research was supported by the Japan Society for the Promotion of Science (JSPS) through the Funding Program for Next-Generation World-Leading Researchers (NEXT Program), initiated by the Council for Science and Technology Policy (2011/LS003) and a Grant-in-Aid for Research Activity

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Neospora caninum: application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice
Exp Parasitol
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M. Nishimura et al.
Oligomannose-coated liposome-entrapped dense granule protein 7 induces protective immune response to Neospora caninum in cattle
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F. Yarovinsky et al.
Toll-like receptor recognition regulates immunodominance in an antimicrobial CD4+ T cell response
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(2006)
Y. Nishikawa et al.
α2-3 Sialic acid glycoconjugate loss and its effect on infection with Toxoplasma parasites
Exp Parasitol
(2013)
R.M. Martin et al.
The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice
J Immunol Methods
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J.L. Jones et al.
Congenital toxoplasmosis: a review
Obstet Gynecol Surv
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Parasite Immunol
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Immunization with oligomannose-coated liposome-entrapped dense granule protein7 protects dams and offspring from Neospora caninum infection in mice
Clin Vaccine Immunol
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Molecular characterization of a profilin gene from a parasitic ciliate Cryptocaryon irritans
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Citation Excerpt :
For example, the immunization of the recombinant profilins from 3 babesia species expressed in E. coli induced cross-protective immunity against B. microti infection in BALB/c mice, characterized by high levels of cytokines and immunoglobulin (IgG) titers, a reduction in peak parasitemia levels, and earlier clearance of the parasite as compared with control mice (Munkhjargal et al., 2016). The profilin of T. gondii formulated with oligomannose-coated liposomes induced high titers of IgG2a and interferon gamma (IFN-γ) in C57BL/6, increasing the survival of infected mice and reducing the parasite burden in their brains (Tanaka et al., 2014). Transgenic Eimeria tenella expressing profilin of Eimeria maxima elicits increased numbers of IFN-γ-secreting lymphocytes and better protective immunity against E. tenella challenge than the wild type (Tang et al., 2018).
Profilin, known as one of the core actin-binding proteins, is an integral part of actin-based cytoskeleton involved in cell motility, cytokinesis, neuronal differentiation, and synaptic plasticity. In this study, a putative profilin gene designated as CiProfilin (GenBank accession number: JX987286) was screened out from a cDNA library of Cryptocaryon irritans trophonts. The full-length cDNA of CiProfilin gene is 582 bp, containing an open reading frame (ORF) of 471 bp, which encodes a polypeptide consisting of 156 amino acids with a predicted molecular weight of 17.3 kDa. Quantification of CiProfilin mRNA expression by real-time PCR suggested that CiProfilin was expressed in all stages of C. irritans life cycle with a significantly higher level in trophonts. Five non-universal codons (TAAs) coding glutamines (Gln) were found in the ORF and mutated to CAAs (universal codons for Gln) by site-directed mutagenesis. Then the modified ORF was inserted into the plasmid pGEX-4T-1, the recombinant plasmid was subsequently transformed into Escherichia coli. The bacteria were subsequently induced to express the recombinant CiProfilin protein fused with glutathione S transferase (G-rCiProfilin), which was then purified with glutathione sepharose 4B and thrombin cleavage. The molecular weight and the antigenicity of rCiProfilin were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The native CiProfilin was found abundant in the peripheral area beneath the cell membrane and around the cytostomes of theronts, suggesting its vital roles in food uptake, stomatogenesis, and parasitic invasion. Co-precipitation assay also revealed the activity of rCiProfilin in actin binding. This study will help further elucidate the specific roles of CiProfilin on the growth of C. irritans and the preliminary mechanism of its invasion to hosts.
Synergistic effect of GRA7 and profilin proteins in vaccination against chronic Toxoplasma gondii infection
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Citation Excerpt :
In other studies where a recombinant profilin was used, a significant humoral response was elicited in CBA/J mice immunized without adjuvant [33], while in C57BL/6 mice no response was observed when the protein was administered alone [17]. However, when encapsulated in liposomes it triggered a Th1 response [17]. On the other hand, vaccination with a DNA vaccine encoding TgPF induced specific Th1 humoral responses in Kunming mice [18,19].
Toxoplasmosis is a zoonotic disease with worldwide prevalence in humans and warm-blooded animal populations. In livestock Toxoplasma gondii is the causal agent of significant economic losses since it can cause abortions in goats and sheep. It is estimated that one third of the world population is infected. Although there are effective therapies for acute infection, these are sometimes poorly tolerated, teratogenic, and have a long administration time. Considering the deficiencies that exist related to the prevention and treatment of toxoplasmosis, the development of a safe and effective vaccine would be extremely valuable in fighting against this infection. In the present work, we characterize for the first time the adjuvant and immunogenic potential of a recombinant profilin protein (rTgPF), in a vaccine formulation alone or in combination with the well-known GRA7 antigen candidate in a murine toxoplasmosis model. Since TgPF acts as a ligand for TLR11 and 12 inducing innate immune responses that promote type 1 adaptive responses, we first study the capacity of the mix rGRA7 + rTgPF to initiate an immune response by evaluating dendritic cell activation. Both rTgPF and rGRA7 induces activation of mouse BMDCs more efficiently than the single proteins, evidenced by increased expression of CD80 and CD86 co-stimulatory proteins and secretion of IL-6, IL-10 and IL-12 cytokines after in vitro stimulation. The sum of the effects of rGRA7 and rTgPF on BMDCs maturation led us to assay them in a vaccination protocol. BALB/c mice vaccinated with this mix elicited a Th1-biased immunity via the induction of lymphocyte proliferation, activation of CD4⁺T cells and increased IFN-γ production that resulted in enhanced protection against chronic Toxoplama gondii infection. Profilin per se induce only cellular immunity but augments the effect of rGRA7 immune responses when used together, thus allowing us to postulate rTgPF as a potential adjuvant in a protein vaccine.
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Since the discovery of Toxoplasma gondii in 1908, it is estimated that one-third of the global population has been exposed to this ubiquitous intracellular protozoan. The complex life cycle of T. gondii has enabled itself to overcome stress and transmit easily within a broad host range thus achieving a high seroprevalence worldwide. To date, toxoplasmosis remains one of the most prevalent HIV-associated opportunistic central nervous system infections. This review presents a comprehensive overview of different vaccination approaches ranging from traditional inactivated whole-T. gondii vaccines to the popular DNA vaccines. Extensive discussions are made to highlight the challenges in constructing these vaccines, selecting adjuvants as well as delivery methods, immunisation approaches and developing study models. Herein we also deliberate over the latest and promising enhancement strategies that can address the limitations in developing an effective T. gondii prophylactic vaccine.
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Toxoplasmosis is an infectious disease caused by the intracellular parasite Toxoplasma gondii. Although almost 1/3 of the world’s population are seropositive, there is no effective vaccine against toxoplasmosis. Therefore, the development of an effective vaccine for control of toxoplasmosis is one of major concerns in parasitology. The aim of this study was to evaluate the efficacy of nano-liposomal excretory-secretory antigens (NLESA) in BALB/c mice.
Excretory-secretory antigens (ESA) was obtained from tachyzoites, encapsulated in the liposome and studied by scanning electron microscope. BALB/c mice were immunized with NLESA and ESA, sterile phosphate-buffered saline (PBS). Immunization was performed three times at 14-day intervals and challenged with 1 × 10⁴ tachyzoites of T. gondii RH strain four weeks later. The parasite load of mice blood, brain and spleen tissues were determined using quantitative PCR targeted at the repeated element (RE) gene.
The immunization with NLESA and ESA induced a significant increase of anti-Toxoplasma IgG antibody compared with PBS group (P < 0.05). After challenge with tachyzoites, qPCR analyses showed significant reduction of parasite load in NLESA and ESA immunized mice compared with control group (P < 0.05). Also, NLESAs were more effective than ESAs and showed significantly reduced parasite load in blood (P = 0.001) and brain tissue (P = 0.01).
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Vaccine

Highlights

Abstract

Introduction

Section snippets

Mice and parasites

Preparation of recombinant protein and liposomes

Immunogenicity of TgPF in Toxoplasma gondii-infected mice

Survival rates of immunized mice after challenge with Toxoplasma gondii

Discussion

Conflicts of interest statement

Acknowledgments

References (16)

Neospora caninum: application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice

Exp Parasitol

Oligomannose-coated liposome-entrapped dense granule protein 7 induces protective immune response to Neospora caninum in cattle

Vaccine

Toll-like receptor recognition regulates immunodominance in an antimicrobial CD4+ T cell response

Immunity

α2-3 Sialic acid glycoconjugate loss and its effect on infection with Toxoplasma parasites

Exp Parasitol

The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice

J Immunol Methods

Congenital toxoplasmosis: a review

Obstet Gynecol Surv

Intraperitoneal immunization with oligomannose-coated liposome-entrapped soluble leishmanial antigen induces antigen-specific T-helper type immune response in BALB/c mice through uptake by peritoneal macrophages

Parasite Immunol

Immunization with oligomannose-coated liposome-entrapped dense granule protein7 protects dams and offspring from Neospora caninum infection in mice

Clin Vaccine Immunol

Cited by (31)

Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen-capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria

Molecular characterization of a profilin gene from a parasitic ciliate Cryptocaryon irritans

Synergistic effect of GRA7 and profilin proteins in vaccination against chronic Toxoplasma gondii infection

Vaccination challenges and strategies against long-lived Toxoplasma gondii

A one health approach to vaccines against Toxoplasma gondii

Quantification of Toxoplasma gondii in the tissues of BALB/c mice after immunization with nanoliposomal excretory-secretory antigens using Real-Time PCR

Short communicationVaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii

Highlights

Abstract

Introduction

Section snippets

Mice and parasites

Preparation of recombinant protein and liposomes

Immunogenicity of TgPF in Toxoplasma gondii-infected mice

Survival rates of immunized mice after challenge with Toxoplasma gondii

Discussion

Conflicts of interest statement

Acknowledgments

Exp Parasitol

Vaccine

Immunity

Exp Parasitol

J Immunol Methods

Congenital toxoplasmosis: a review

Obstet Gynecol Surv

Intraperitoneal immunization with oligomannose-coated liposome-entrapped soluble leishmanial antigen induces antigen-specific T-helper type immune response in BALB/c mice through uptake by peritoneal macrophages

Parasite Immunol

Immunization with oligomannose-coated liposome-entrapped dense granule protein7 protects dams and offspring from Neospora caninum infection in mice

Clin Vaccine Immunol

Short communication
Vaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii