• Parasites, Hosts and Diseases
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• v.56 no.5
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• pp.429-435
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• 2018

Toxoplasma gondii is a ubiquitous protozoan parasite responsible for causing toxoplasmosis. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines are of urgent need. In this study, we generated 2 virus-like particles (VLPs) vaccines expressing T. gondii rhoptry protein 4 (ROP4) or rhoptry protein 18 (ROP18) using influenza matrix protein (M1) as a core protein. Mice were intranasally immunized with VLPs vaccines and after the last immunization, mice were challenged with ME49 cysts. Protective efficacy was assessed and compared by determining serum antibody responses, body weight changes and the reduction of cyst counts in the brain. ROP18 VLPs-immunized mice induced greater levels of IgG and IgA antibody responses than those immunized with ROP4 VLPs. ROP18 VLPs immunization significantly reduced body weight loss and the number of brain cysts in mice compared to ROP4 VLPs post-challenge. These results indicate that T. gondii ROP18 VLPs elicited better protective efficacy than ROP4 VLPs, providing important insight into vaccine design strategy.

• Clinical and Experimental Vaccine Research
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• v.13 no.4
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• pp.359-369
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• 2024

The present study aimed to evaluate the key characteristics of Toxoplasma gondii rhoptry protein 4 (TgROP4), including physicochemical parameters, structural features, immunogenic epitopes, and virtual immune simulation, using several bioinformatics-based servers and tools. Based on allergenicity and antigenicity outputs, the TgROP4 protein seemed to have an immunogenic and non-allergenic nature. The quality of the three-dimensional (3D) structure improved after refinement, according to the outcomes of the Ramachandran plot and the ProSA-web servers. ABCpred and SVMTriP web tools were used to predict linear B lymphocyte epitopes and found several promising epitopes. Acceptable antigenicity, hydrophilicity, beta-turn, Bepipred linear epitope 2.0, flexibility, and surface accessibility scores were obtained through the Immune Epitope Database (IEDB). Also, seven discontinuous B-cell epitopes ranging from scores 0.966 to 0.848 were found in the 3D model of TgROP4 via the ElliPro. The IEDB findings showed T-cell epitopes on TgROP4 protein are capable to strongly bind to the major histocompatibility complex classes. In silico immune simulation was performed using C-ImmSim server and showed three injections of TgROP4 protein at 4-week intervals is capable to elicit adequate humoral and cell-mediated immune responses.

• Parasites, Hosts and Diseases
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• v.57 no.5
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• pp.543-547
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• 2019

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 na?ve control mice perished. These results indicate that the ROP13 VLPs vaccine could be a potential vaccine candidate against T. gondii infection.

• Parasites, Hosts and Diseases
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• v.55 no.2
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• pp.143-148
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• 2017

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.

• Parasites, Hosts and Diseases
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• v.59 no.6
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• pp.565-572
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• 2021

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.

• Parasites, Hosts and Diseases
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• v.37 no.3
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• pp.157-162
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• 1999

Serum from mouse orally ingested with tissue cyst forming stain (Me49) of Toxoplasma gondii was assayed by Western blot and immunofluorescene assay (IFA) to establish early responses in antigenicity of the parasite in mouse model of foodborne toxoplasmosis. Sera were collected weekly to blot the RH antigen transferred onto nitrocellulose paper after being separated by 12% SDS-PAGE. With the second week serum, 34 kDa protein (p34) was detected uniquely, and all antigens of T.gondii were detected with the sera from 3 or 4 weeks. p34 was not a member of the major surface membrane proteins and confirmed to be localized in the rhoptry by IFA. It was secreted into parasitophorous vacuolar membrane (PVM) during the entry into host cells. 10.3% of sera detected p34, while all the ELISA positive sera detected the band. It has diagnostic usefulness of presumed T.gondii infection. We suggest the name of the p34 protein as ROP9.

• Parasites, Hosts and Diseases
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• v.52 no.4
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• pp.439-441
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• 2014

Toxoplasma gondii is the causative agent of toxoplasmosis with symptoms of congenital neurological and ocular diseases and acquired lymphadenitis, retinochoroiditis, and meningoencephalitis. Small molecules which block the activity of protein kinases were tested in in vitro culture of T. gondii to find new therapeutic drugs of safer and more effective than the combined administration of pyrimethamine and sulfadoxine that sometimes provoke lethal Stevens-Johnson syndrome. Among them, Gefitinib and Crizotinib inhibited intracellular growth of T. gondii in HeLa cells by counting the number of T. gondii per parasitophorous vacuolar membrane whereas Sunitinib did not. Gefitinib inhibited the growth of T. gondii in a dose-dependent manner over $5{\mu}M$ up to the tolerable concentration of HeLa cells and halted the division of the parasite immediately from the time point of treatment. Gefitinib inhibition suggests that tyrosine kinases of EGFR family or other homologous kinases of the parasite itself may be the target to cause the block of T. gondii growth.