Immunization of C57BL/6 Mice with GRA2 Combined with MPL Conferred Partial Immune Protection against Toxoplasma gondii

Background: We have previously reported that immunization with GRA2 antigen of Toxoplasma gondii induces protective immunity in CBA/J (H2k) and BALB/c mice (H2d). We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens (GRA2) combined with monophosphorryl lipid A (MPL) adjuvant elicits protective immune response against T. gondii. Methods: C57BL/6 (H2b haplotype) mice were immunized with GRA2, formulated in MPL adjuvant. Results: Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-γ, was detected at three weeks post immunization. Mice immunized with GRA2 had significantly (p < 0.01) fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-γ production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization. Conclusion: We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA2 for incorporation into a multi-antigenic vaccine against T. gondii.

SHuffle™ T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties.

BACKGROUND: Human fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1. METHODS: A synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts (BL21 (DE3), Rosetta-gami™ 2(DE3), SHuffle™ T7). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties. RESULTS: The highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84ng/mL. CONCLUSION: Although SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.

Synergistic effect of rSAG1 and rGRA2 antigens formulated in PLGA microspheres in eliciting immune protection against Toxoplasama gondii.

There is still no human vaccine against Toxoplasma gondii (T. gondii), as one of the most successful parasites. In present study, we designed a subunit vaccine composed of recombinant SAG1 (rSAG1) and recombinant GRA2 (rGRA2) proteins. In order to improve the induced immune responses, rSAG1 and rGRA2 were adsorbed on Poly (DL-lactide-co-glycolide) (PLGA) microspheres (MS) prepared by double emulsion solvent evaporation method. BALB/c mice were subcutaneously vaccinated by rSAG1-adsorbed PLGA MS (rSAG1-PLGA), rGRA2-adsorbed PLGA MS (rGRA2-PLGA), and the mixture of both formulations (rSAG1/rGRA2-PLGA), twice with a 3-week interval. PLGA MS characteristics, protein release, cellular and humoral immune responses, and protection against acute toxoplasmosis were evaluated. All vaccinated mice induced significantly partial protection and longer survival times associated with higher IFN-γ/IL-10 ratio and higher amount of Toxoplasma-specific IgG antibodies compared to control groups. Interestingly, the synergistic effect of rSAG1 and rGRA2 in eliciting more potent cellular and humoral responses and consequently higher protection in comparison to single antigen was confirmed. This study introduces the mixture of rSAG1 and rGRA2 (derived from different stages of Toxoplasma life-cycle) formulated in PLGA MS as a promising candidate in vaccine development against T. gondii.

Factors associated with body image dissatisfaction and distortion among Iranian women.

The prevalence of body image dissatisfaction is considered high in both developed and developing countries. It has been shown that many factors affect the body image dissatisfaction. However, because of the economical and cultural differences, it seems that these affecting factors should be determined in each region. So, the present study was designed to evaluate the prevalence and associated factors with body image dissatisfaction and distortion among Iranian women. Body image perception was analyzed in 500 women through the Stunkard figure rating system. The International Physical Activity Questionnaire was used for assessing physical activity level. The information about age, marital status and socioeconomic status was recorded by general questionnaire. The one-way ANOVA and regression were used for statistical analysis. By increasing the BMI categories from underweight to obesity, participants tended to perceive their body size thinner than real body size. The regression models indicated that the body image distortion was significantly increased with increasing the BMI (p=0.002) and physical activity level (p=0.008). Besides, dissatisfaction by being heavier than ideal was significantly associated with higher BMI (OR (95% CI). 1.21 (1.03, 1.17)). Considering the high prevalence of body image dissatisfaction among Iranian women, for preventing psychological problems and eating disorders, appropriate public health programs for increasing awareness about healthy body size were needed.

Production of in-vitro refolded and highly antigenic SAG1 for development of a sensitive and specific Toxoplasma IgG ELISA.

Recombinant antigens are increasingly applied to replace native antigens in serological tests. Surface antigen 1 (SAG1) is a highly immunogenic antigen and probably represents the most explored and used antigen of Toxoplasma gondii for development of serological test kits. The presence of six disulfide bridges in its structure makes SAG1 a highly conformational protein. In fact, antigenicity of SAG1 is greatly dependent on proper disulfide bonding and folding. In-vitro refolding of SAG1 inclusion bodies, produced in Escherichia coli, was reported to result in soluble and antigenic protein. We produced SAG1 in E. coli and highly purified it by a single denaturing immobilized metal affinity chromatography. Refolding of denatured SAG1 was performed by (a) dialysis in the presence of reduced/oxidized glutathione, (b) drop-wise dilution and (c) drop-wise dilution in the presence of CuSo4. Refolding in the presence of oxido-shuffling reagent was much more efficient in producing presumably correctly-folded and highly antigenic SAG1 as demonstrated by non-reducing SDS-gel electrophoresis, ELISA, Western blotting and reversed-phase HPLC. An IgG ELISA developed using SAG1 refolded in the presence of oxido-shuffling reagent displayed high sensitivity and specificity for detection of Toxoplasma IgG antibodies in pregnant women.

Expression and Purification of Recombinant ROP1 of Toxoplasma gondii in Bacteria.

BACKGROUND: Toxoplasmosis is a worldwide-distributed infection which is mostly asymptomatic but can cause serious health problems in congenitally-infected newborns and immunecompromised individuals. Research is undergoing both to improve Toxoplasma serological tests, which play the main role in laboratory diagnosis of the infection, and develop an effective vaccine to prevent the infection. Some studies showed usefulness of rhoptry protein 1 (ROP1) antigen of Toxoplasma gondii (T. gondii) in serodiagnosis of the infection and induction of protective immunity. The purpose of this study was to produce recombinant ROP1 and evaluate its antigenicity against human infected sera. METHODS: DNA encoding ROP1, amino acids 171 to 574, was obtained from T. gondii RH strain by polymerase chain reaction amplification and cloned in prokaryotic expression plasmid pET-15b. rROP1 was expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography. RESULTS: DNA sequencing showed 99% similarity between the cloned sequence and the corresponding sequence in Gene bank. Results indicated the proper antigenicity of rROP1. Sera from Toxoplasma infected individuals specifically recognized rROP1 in Western blotting. CONCLUSION: rROP1 is antigenic toward human infected sera and can be used in studies for development of both a Toxoplasma serological test and a protective vaccine.

Seroprevalence and risk factors for Toxoplasma gondii infection among pregnant women in Northeast Iran.

We report Toxoplasma IgG seroprevalence of 34.4% among 419 pregnant women in Mashhad, northeast Iran. Soil contact, living in rural environment, and level of education were associated with infection. The prevalence did not increase with age, suggesting high infection rate during childhood and adolescence.