Immunoinformatic analysis of immunogenic B- and T-cell epitopes of MIC4 protein to designing a vaccine candidate against Toxoplasma gondii through an in-silico approach

Purpose@#Toxoplasmosis, transmitted by Toxoplasma gondii, is a worldwide parasitic disease that affects approximately one-third of the world’s inhabitants. Today, there are no appropriate drugs to deter tissue cysts from developing in infected hosts. So, developing an effective vaccine would be valuable to avoid from toxoplasmosis. Considering the role of microneme antigens such as microneme protein 4 (MIC4) in T. gondii pathogenesis, it can be used as potential candidates for vaccine against T. gondii. @*Materials and Methods@#In this study several bioinformatics methods were used to assess the different aspects of MIC4 protein such as secondary and tertiary structure, physicochemical characteristics, the transmembrane domains, subcellular localization, B-cell, helper-T lymphocyte, cytotoxic-T lymphocyte epitopes, and other notable characteristic of this protein design a suitable vaccine against T. gondii. @*Results@#The studies revealed that MIC4 protein includes 59 potential post-translational modification sites without any transmembrane domains. Moreover, several probable epitopes of Band T-cells were detected for MIC4. The secondary structure comprised 55.69% random coil, 5.86% beta-turn, 19.31% extended strand, and 19.14% alpha helix. According to the Ramachandran plot results, 87.42% of the amino acid residues were located in the favored, 9.44% in allowed, and 3.14% in outlier regions. The protein allergenicity and antigenicity revealed that it was non-allergenic and antigenic. @*Conclusion@#This study gives vital basic on MIC4 protein for further research and also established an effective vaccine with different techniques against acute and chronic toxoplasmosis.

Antigenic properties of dense granule antigen 12 protein using bioinformatics tools in order to improve vaccine design against Toxoplasma gondii

Purpose@#Toxoplasma gondii is an opportunistic parasite infecting all warm-blooded animals including humans. The dense granule antigens (GRAs) play an important role in parasite survival and virulence and in forming the parasitophorous vacuole. Identification of protein characteristics increases our knowledge about them and leads to develop the vaccine and diagnostic studies. @*Materials and Methods@#This paper gave a comprehensive definition of the important aspects of GRA12 protein, including physico-chemical features, a transmembrane domain, subcellular position, secondary and tertiary structure, potential epitopes of B-cells and T-cells, and other important features of this protein using different and reliable bioinformatics methods to determine potential epitopes for designing of a high-efficient vaccine. @*Results@#The findings showed that GRA12 protein had 53 potential post-translational modification sites. Also, only one transmembrane domain was recognized for this protein. The secondary structure of GRA12 protein comprises 35.55% alpha-helix, 19.50% extended strand, and 44.95% random coil. Moreover, several potential B- and T-cell epitopes were identified for GRA12. Based on the results of the Ramachandran plot, 79.26% of amino acid residues were located in favored, 11.85% in allowed and 8.89% in outlier regions. Furthermore, the results of the antigenicity and allergenicity assessment noted that GRA12 is immunogenic and nonallergenic. @*Conclusion@#This research provided important basic and conceptual data on GRA12 to develop an effective vaccine against acute and chronic toxoplasmosis for further in vivo investigations. More studies are required on vaccine development using the GRA12 alone or combined with other antigens in the future.

Antigenic properties of dense granule antigen 12 protein using bioinformatics tools in order to improve vaccine design against Toxoplasma gondii

Purpose@#Toxoplasma gondii is an opportunistic parasite infecting all warm-blooded animals including humans. The dense granule antigens (GRAs) play an important role in parasite survival and virulence and in forming the parasitophorous vacuole. Identification of protein characteristics increases our knowledge about them and leads to develop the vaccine and diagnostic studies. @*Materials and Methods@#This paper gave a comprehensive definition of the important aspects of GRA12 protein, including physico-chemical features, a transmembrane domain, subcellular position, secondary and tertiary structure, potential epitopes of B-cells and T-cells, and other important features of this protein using different and reliable bioinformatics methods to determine potential epitopes for designing of a high-efficient vaccine. @*Results@#The findings showed that GRA12 protein had 53 potential post-translational modification sites. Also, only one transmembrane domain was recognized for this protein. The secondary structure of GRA12 protein comprises 35.55% alpha-helix, 19.50% extended strand, and 44.95% random coil. Moreover, several potential B- and T-cell epitopes were identified for GRA12. Based on the results of the Ramachandran plot, 79.26% of amino acid residues were located in favored, 11.85% in allowed and 8.89% in outlier regions. Furthermore, the results of the antigenicity and allergenicity assessment noted that GRA12 is immunogenic and nonallergenic. @*Conclusion@#This research provided important basic and conceptual data on GRA12 to develop an effective vaccine against acute and chronic toxoplasmosis for further in vivo investigations. More studies are required on vaccine development using the GRA12 alone or combined with other antigens in the future.

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.

Calcium-dependent protein kinases are potential targets for Toxoplasma gondii vaccine

Toxoplasma gondii belongs to the Apicomplexa phylum that caused a widespread zoonotic infection in wide range of intermediate hosts. Over one-third of the world's population are latently infected with T. gondii and carry it. The complex life cycle of T. gondii indicates the presence of a plurality of antigenic epitopes. During the recent years, continuous efforts of scientists have made precious advances to elucidate the different aspects of the cell and molecular biology of T. gondii. Despite of great progresses, the development of vaccine candidates for preventing of T. gondii infection in men and animals is still remains a challenge. The calcium-dependent protein kinases (CDPKs) belongs to the superfamily of kinases, which restricted to the apicomplexans, ciliates, and plants. It has been documented that they contribute several functions in the life cycle of T. gondii such as gliding motility, cell invasion, and egress as well as some other critical developmental processes. In current paper, we reviewed the recent progress concerning the development of CDPK-based vaccines against acute and chronic T. gondii.

Recent progress in microneme-based vaccines development against Toxoplasma gondii

Toxoplasmosis is a cosmopolitan zoonotic disease, which infect several warm-blooded mammals. More than one-third of the human population are seropositive worldwide. Due to the high seroprevalence of Toxoplasma gondii infection worldwide, the resulting clinical, mental, and economical complications, as well as incapability of current drugs in the elimination of parasites within tissue cysts, the development of a vaccine against T. gondii would be critical. In the past decades, valuable advances have been achieved in order to identification of vaccine candidates against T. gondii infection. Microneme proteins (MICs) secreted by the micronemes play a critical role in the initial stages of host cell invasion by parasites. In this review, we have summarized the recent progress for MIC-based vaccines development, such as DNA vaccines, recombinant protein vaccines, vaccines based on live-attenuated vectors, and prime-boost strategy in different mouse models. In conclusion, the use of live-attenuated vectors as vehicles to deliver and express the target gene and prime-boost regimens showed excellent outcomes in the development of vaccines against toxoplasmosis, which need more attention in the future studies.

DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice

Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis.The aim of the present study was to compare the immune responses induced following DNA vaccinationwith LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genesalone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immuneresponses were evaluated before and after challenge with Leishmania major (L. major). In addition,the mean lesion size was also measured from 3th week post-infection. All immunized mice showed apartial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levelscompared to control groups (p<0.05). IFN-γ/ Interleukin (IL)-4 and IgG2a/IgG1 ratios demonstratedthe highest IFN-γ and IgG2a levels in the group receiving LACK–TSA fusion. Mean lesion sizesreduced significantly in all immunized mice compared with control groups at 7th week post-infection(p<0.05). In addition, there was a significant reduction in mean lesion size of LACK-TSA andTSA groups than LACK group after challenge (p<0.05). In the present study, DNA immunizationpromoted Th1 immune response and confirmed the previous observations on immunogenicity ofLACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccinecan induce stronger immune responses and protection against infectious challenge with L. major.

Evaluation of protective effect of IL-22 and IL-12 on cutaneous leishmaniasis in BALB/c mice

Objective: To investigate the protective effect of IL-22 and IL-12 on cutaneous leishmaniasisin BALB/c mice. Methods: The protective effect of IL-22 and IL-12 on cutaneous leishmanias in BALB/c mice was evaluated by measurement of IL-4, INF-γ, total IgG, IgG1 and IgG2a after challenge with Leishamania major. Clinical evaluations were performed by measurement of lesion diameter, and survival rate of the mice. Results: In week 27 post infection, the mortality rates for control groups were 100%. While the survival rates for the IL-12, IL-12 + IL-22, and IL-22(5 ng/g) groups were 100%. The size of lesions decreased in the presence IL-22 (5 ng/g) of mice weight, which was statistically significant in comparison with other groups (. P<0.05). Mean of total IgG, IgG1 and IgG2a for IL-22 (5 ng/g) group was more than other groups. In IL-22 group (5 ng/g), INF-γ production was significantly higher than other groups and IL-4 was significantly lower than other groups. Conclusions: The results obtained indicate the effectiveness of IL-22 and its effect on IL-12 in protection of cutaneous leishmaniasis.

Evaluation of protective effect of IL-22 and IL-12 on cutaneous leishmaniasis in BALB/c mice

OBJECTIVE@#To investigate the protective effect of IL-22 and IL-12 on cutaneous leishmaniasisin BALB/c mice.@*METHODS@#The protective effect of IL-22 and IL-12 on cutaneous leishmanias in BALB/c mice was evaluated by measurement of IL-4, INF-γ, total IgG, IgG1 and IgG2a after challenge with Leishamania major. Clinical evaluations were performed by measurement of lesion diameter, and survival rate of the mice.@*RESULTS@#In week 27 post infection, the mortality rates for control groups were 100%. While the survival rates for the IL-12, IL-12 + IL-22, and IL-22(5 ng/g) groups were 100%. The size of lesions decreased in the presence IL-22 (5 ng/g) of mice weight, which was statistically significant in comparison with other groups (P<0.05). Mean of total IgG, IgG1 and IgG2a for IL-22 (5 ng/g) group was more than other groups. In IL-22 group (5 ng/g), INF-γ production was significantly higher than other groups and IL-4 was significantly lower than other groups.@*CONCLUSIONS@#The results obtained indicate the effectiveness of IL-22 and its effect on IL-12 in protection of cutaneous leishmaniasis.

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

BACKGROUND: TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. METHODS: Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. CONCLUSION: The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania major and Expression in the Chinese Hamster Ovary Cell Line

Background: TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Methods: Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting. Results: The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. Conclusion: The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.