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

Abstract

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_HC) in a T. gondii infection murine model.

Methods: In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1_HC fusion protein (NbHsp90.3-SAG1_HC 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_HC-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_TLA) 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_HC-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_HC group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1_HC-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_HC 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.

Keywords: Adjuvant; Epitopes; Plant Hsp90; SAG1; Toxoplasma gondii; Toxoplasmosis; Vaccine.

Fig. 1

Levels of anti-SAG1 total IgG antibodies in the sera of C57BL/6 immunized mice. Two weeks after the last immunization, serum samples (8 mice per group) were collected to analyze IgGt by ELISA with rSAG1m (a) and SAG1_TLA (b) as the bound target. Sera were diluted 1:1000 or 1:100 for detecting rSAG1m or SAG1_TLA, respectively. Each bar represents the group mean ± SEM. Results represent one of three similar experiments. Pre-immune sera were used as a negative control. a ****P < 0.0001, NbHsp90.3_HC vs PBS, NbHsp90.3 and SAG1m groups. b *P < 0.05, NbHsp90.3_HC vs SAG1m group; ***P < 0.001, NbHsp90.3_HC vs PBS and NbHsp90.3 groups. Statistical analysis was performed by one-way analysis of variance (ANOVA) using Tukey’s multiple comparisons test

Fig. 2

Analysis of IgG isotype antibodies against SAG1 in the sera of C57BL/6 immunized mice. Two weeks after the immunization schedule was completed, serum samples (8 mice per group) were collected and diluted 1:100 to analyze IgG1, IgG2a and IgG2b sub-classes with rSAG1m (a) or SAG1_TLA (b) as the bound target. Each bar represents the group mean ± SEM. Results represent one of three similar experiments. Pre-immune sera were used as a negative control. *P < 0.05; ***P < 0.001; ****P < 0.0001. Statistical analysis was performed by two-way analysis of variance (ANOVA) using Tukey’s multiple comparisons test

Fig. 3

Cytokine production by spleen cell cultures from C57BL/6 immunized mice. Two weeks after last immunization, splenocytes from 5 mice per group were cultured, and the cytokine production in cell supernatants after rSAG1 stimulation was measured by ELISA. Values for IFN-γ (a) and IL-10 (b) at 72 h are expressed as mean ± SEM. Results represent one of two similar experiments. IFN-γ: ***P < 0.001 NbHsp90.3-SAG1_HC vs PBS, NbHsp90.3 and SAG1m groups. IL-10: *P < 0.05, NbHsp90.3 vs NbHsp90.3-SAG1_HC group; **P < 0.01, NbHsp90.3 vs PBS group; ***P < 0.001, SAG1m vs NbHsp90.3-SAG1_HC group; ****P < 0.0001 SAG1m vs PBS group. Statistical analysis was performed by two-way analysis of variance (ANOVA) using Tukey’s multiple comparisons test

Fig. 4

Post-challenge analysis in C57BL/6 immunized mice after T. gondii infection. a Protection against chronic T. gondii infection in C57BL/6 immunized mice. Two weeks after the last immunization, mice were orally challenged with 20 tissue cysts of ME49 T. gondii strain (sub-lethal dose). Thirty days after the infection, mice were sacrificed, and their brains were removed for cyst load determination. Each bar represents the group mean ± SEM. Results represent one of three similar experiments. **P < 0.01, NbHsp90.3-SAG1_HC vs NbHsp90.3 group; ****P < 0.0001, NbHsp90.3-SAG1_HC vs PBS and SAG1m groups. b, c Antigen-specific antibodies in sera from C57BL/6 immunized mice with chronic T. gondii infection. Thirty days after mice were orally challenged with 20 tissue cysts of ME49 T. gondii strain (sub-lethal dose), serum samples (8 mice per group) were obtained and IgGt was determined by ELISA with TLA (b) or rSAG1m (c) as the bound targets (sera were diluted 1:16,000 and 1:1000, respectively). Each bar represents the group mean ± SEM. Results represent one of two similar experiments. b ***P < 0.001, NbHsp90.3-SAG1_HC vs PBS, NbHsp90.3 and SAG1m groups. c **P < 0.01, NbHsp90.3-SAG1_HC vs SAG1m group; ***P < 0.001, NbHsp90.3-SAG1_HC vs PBS and NbHsp90.3 groups. Pre-immune sera were used as a negative control. Statistical analysis was performed by one-way analysis of variance (ANOVA) using Tukey’s multiple comparisons test

Fig. 5

Cytokine detection in spleen cell cultures and sera from C57BL/6 immunized mice after T. gondii infection. a Values of IFN-γ in serum samples (5 mice per group) at 10 days (acute infection) and 30 days (chronic infection) after mice were orally challenged with 20 tissue cysts of ME49 T. gondii strain (sub-lethal dose). Sera were diluted 1:10 and IFN-γ was measured by ELISA. Values of IFN-γ (b) and IL-10 (c) in supernatants of spleen cell cultures in the chronic infection. Splenocytes from 5 mice per group were cultured and stimulated with either rSAG1 or rGRA4. Cytokine was measured in cell supernatants after 72 h of culture by ELISA. Each bar represents the group mean ± SEM. Results represent one of two similar experiments. IFN-γ: *P < 0.05, NbHsp90.3-SAG1_HC vs PBS, NbHsp90.3 and SAG1m groups. IL-10: ***P < 0.001, NbHsp90.3-SAG1_HC vs PBS, NbHsp90.3 and NbHsp90.3-SAG1_HC groups. Statistical analysis was performed by two-way analysis of variance (ANOVA) using Tukey’s multiple comparisons test