Rhoptry antigens as Toxoplasma gondii vaccine target

Toxoplasmosis is a cosmopolitan zoonotic infection, caused by a unicellular protozoan parasite known as Toxoplasma gondii that belongs to the phylum Apicomplexa. It is estimated that over one-third of the world's population has been exposed and are latently infected with the parasite. In humans, toxoplasmosis is predominantly asymptomatic in immunocompetent persons, while among immunocompromised individuals may be cause severe and progressive complications with poor prognosis. Moreover, seronegative pregnant mothers are other risk groups for acquiring the infection. The life cycle of T. gondii is very complex, indicating the presence of a plurality of antigenic epitopes. Despite of great advances, recognize and construct novel vaccines for prevent and control of toxoplasmosis in both humans and animals is still remains a great challenge for researchers to select potential protein sequences as the ideal antigens. Notably, in several past years, constant efforts of researchers have made considerable advances to elucidate the different aspects of the cell and molecular biology of T. gondii mainly on microneme antigens, dense granule antigens, surface antigens, and rhoptry proteins (ROP). These attempts thereby provided great impetus to the present focus on vaccine development, according to the defined subcellular components of the parasite. Although, currently there is no commercial vaccine for use in humans. Among the main identified T. gondii antigens, ROPs appear as a putative vaccine candidate that are vital for invasion procedure as well as survival within host cells. Overall, it is estimated that they occupy about 1%–30% of the total parasite cell volume. In this review, we have summarized the recent progress of ROP-based vaccine development through various strategies from DNA vaccines, epitope or multi epitope-based vaccines, recombinant protein vaccines to vaccines based on live-attenuated vectors and prime-boost strategies in different mouse models.

[Evaluation of immunization of BALB/c mice by plasmid encoding complete ROP2 gene of Toxoplasma gondii]

Toxoplasma gondii is an obligate intracellular protozoan that causes Toxoplasmosis in human and animal. In recent years, significant progress has been made in the identification of vaccine candidates which can induce protective responses. In this study we used complete Rhoptry protein 2 gene of Toxoplasma gondii as a single DNA vaccine and evaluated its immune responses in comparison with control groups. BALB/c mice were immunized intramuscularly with three weaks time interval with pcROP2 [as case group] and pc-DNA3 and PBS [as control groups]. After immunization, we evaluated the immune response using cytokine and antibody measurements. The results of cytokine [IFN-gamma, IL-4] assays showed that mice immunized with pcROP2, elicited stronger Th1-type cellular immune responses than those immunized with empty plasmid, or PBS [high level of IFN-gamma and low-level of IL-4]. Also Anti-T. gondii IgG titres [OD] increased markedly in the pcROP2 group, which was significantly higher than those of control groups [P<0.05]. When challenged with the highly virulent Toxoplasma gondii RH strain, mice immunized with pcROP2 had siginificantly higher survival rates compared to control groups [P<0.05]. This study showed that pc-ROP2 as a single DNA vaccine is effective to prime enhanced and balanced cellular and humeral immunity responses, and relatively improved mice survival time against toxoplasmosis

production of molecular clones of toxoplasma gondii-derived heat shock protein 70 kDa

Toxoplasmosis is a common and widespread infection in humans and many other species of warmblooded animals. Toxoplasma gondii-derived heat shock protein 70 [Hsp70] may play an important role in the virulence of Toxoplasma gondii [T. gondii]. In the present study, T. gondii Hsp70 was amplified by polymerase chain reaction [PCR] from the DNA of the T. gondiitachyzoite RH strain through the use of specific primers with Xho1 and Xba1 restriction sites. The purified DNA fragment of the T. gondii Hsp70 gene was subcloned into the Xho1 and Xba1-digested eukaryotic expression vector, pcDNA3, and subsequently transformed into TOP10 chemically competent cells. A 2004 base pair [bp] band of PCR product was observed on the 0.8% agarose gel. The cDNA was inserted into the pTZ57R/T vector and then subcloned successfully into the pcDNA3 eukaryotic expression plasmid vector. The sequence of this amplified gene showed up to 100% homology with the target gene according to the Genbank database [Accession no. U82281]

effect of Alkanna tincturia and Peganum harmala extracts on leishmania major [MRHO/IR/75/ER] in vitro

Cutaneous leishmaniasis is an important health problem caused by Leishmania spp. As there is no vaccine, drug treatment is the only way to tackle leishmaniasis. In the present study, inhibitory and killing effects of Peganum harmala and Alkana tinctoria extracts on amastigotes and promastigotes forms of Leishmania were evaluated in-vitro. The seeds of Peganum harmala, Stems and roots of Alkanna tictoria were collected and crude extraction carried out. In this experimental study, Leishmania major promastigotes were cultured in RPMI-1640 with 10% FBS at 22-26°C, and infected macrophages with amastigotes were cultured in RPMI-1640 with 10% FBS at 37°C in 5% CO2. Then the extracts of each plant were added to cultivated parasites and incubated for 3 days. Promastigote and amastigote assay was carried out using counting assay based on growth inhibition. The results indicated that both extractions can inhibit the growth of promastigotes, and in concentrations of 40 micro g/ml of P. harmala, 200 micro g/ml of A. tincturia, and 20 micro g/ml of equal combination of P. hamala and A. tincturia are Inhibitory Concentration [IC50] for parasites growth. By adding these concentrations of the extracts to the infected macrophages in the culture, their effects were separately evaluated. The mean of amastigotes number in macrophages in the culture with P. harmala, A. ticturia, combination and control groups were 0.7, 0.7, 0.6, 2.3 amastigotes per macrophage, respectively. By this method, inhibition of intracellular and extracellular growth of L. major was demonstrated suggesting that, plant drugs with efficacy and safe products can be applied as new treatment for cutaneous leishmaniasis