Plant Hsp90 is a novel adjuvant that elicits a strong humoral and cellular immune response against B- and T-cell epitopes of a Toxoplasma gondii SAG1 peptide.

Background: The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1) in a T. gondii infection murine model.

Methods: In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1 fusion protein (NbHsp90.3-SAG1 group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA.

Results: The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1 group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection.

Conclusions: We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1 exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes.