In vitro expression and seroreactivity of Toxoplasma gondii GRA1 protein encoded by unmodified and...

Toxoplasmosis is an infectious parasitic disease caused by Toxoplasma gondii, with a global seroprevalence estimated to affect more than one-third of the human population. T. gondii can be a serious health concern for pregnant women and immunocompromised patients. Currently, there is no effective and safe vaccine for humans. However, mRNA technology is a promising platform to develop efficient and safe vaccines against toxoplasmosis. Therefore, we designed nucleoside-modified and unmodified mRNA vaccine transcripts expressing T. gondii recombinant GRA1 protein and analyzed their in vitro protein expression profile as well as potential immunogenicity using IFAT, Western blot, and ELISA. Moreover, to compare translational efficiency, poly-A tail was extended with an extra ∼150 bp in addition to the existing ∼120 bp poly-A tail in two distinct mRNA designs. The results showed that all four mRNA constructs expressed rGRA1 protein in HEK293T cells. However, mRNA constructs with extended poly-A tails exhibited reduced expression level of rGRA1 protein compared to other mRNA vaccine transcripts. Additionally, unmodified mRNA construct with or without extended poly-A tail yielded significantly higher protein expression compared to nucleoside-modified mRNA constructs through ELISA using anti-polyhis antibody (P<0.05). Western blot showed that rGRA1 protein expressed by all four mRNA constructs reacted strongly with T. gondii IgG seropositive mice sera. Furthermore, ELISA revealed that rGRA1 proteins derived from unmodified and nucleoside-modified mRNA constructs formed significantly stronger immune complexes with T. gondii IgG antibodies compared to negative controls (P<0.05). These data suggest that both unmodified and nucleosidemodified mRNA constructs expressing rGRA1 protein have potential as vaccine candidates and warrant further investigation for development of an mRNA-based vaccine against toxoplasmosis. However, encapsulation of the produced mRNA constructs in LNPs and in vivo studies are required to validate these preliminary results.