Effective Nanoparticle-Based Nasal Vaccine Against Latent and Congenital Toxoplasmosis in Sheep

Abstract

Toxoplasma gondii is a parasitic protozoan of worldwide distribution, able to infect all warm-blooded animals, but particularly sheep. Primary infection in pregnant sheep leads to millions of abortions and significant economic losses for the livestock industry. Moreover, infected animals constitute the main parasitic reservoir for humans. Therefore, the development of a One-health vaccine seems the best prevention strategy. Following earlier work, a vaccine constituted of total extract of Toxoplasma gondii proteins (TE) associated with maltodextrin nanoparticles (DGNP) was developed in rodents. In this study we evaluated the ability of this vaccine candidate to protect against latent and congenital toxoplasmosis in sheep. After two immunizations by either intranasal or intradermal route, DGNP/TE vaccine generated specific Th1-cellular immune response, mediated by APC-secretion of IFN-γ and IL-12. Secretion of IL-10 appeared to regulate this Th1 response for intradermally vaccinated sheep but was absent in intranasally-vaccinated animals. Finally, protection against latent toxoplasmosis and transplacental transmission were explored. Intranasal vaccination led to a marked decrease of brain cysts compared with the non-vaccinated group. This DGNP/TE vaccine administered intranasally conferred a high level of protection against latent toxoplasmosis and its transplacental transmission in sheep, highlighting the potential for development of such a vaccine for studies in other species.

Keywords: adjuvant free; nanoparticle; nasal vaccination; one-health approach; ovine toxoplasmosis.

FIGURE 1

Vaccination and challenge overview of the latent and the congenital experiments. Experiment day are indicated as numbers (D0-D292). The vaccine schedule is common for both experiments (left): two immunizations 21 days apart (D0, D21), blood sampling for IgG detection by ELISA (D0, D21, and D80), and for PBMC (peripheral blood mononuclear cells) collection for antigen recall assay (D0, D28). In latent experiment (upper right): 6 months after vaccination (D200), spleen and lymph nodes from 2 sheep were used for cellular immune response study, and remaining animals were per os challenged then sacrifice 2 months for brain parasitic load counting. In congenital experiment (lower right): sheep were artificially inseminated 3 months after immunizations (D110), then s.c. challenge (D241) was performed 3 weeks before lambing while protection was evaluated 1 month after lambing by determining the sheep and lamb brain cyst load (D292).

FIGURE 2

Ex vivo tracking of DGNP-DiR administered by intranasal spray or by intradermal inoculation in the neck or in the cheek. DGNP-DiR signal was observed 24 h after administration using the in vivo imaging system IVIS^® Spectrum (PerkinElmer) under epi-illumination with filters set: 745 nm ex/800 nm em (A) Inoculation sites from sheep inoculated with DGNP-DiR by intradermal routes. (B) Sagittal cut of head from sheep inoculated with DGNP-DiR by nasal route A: Brain B: Turbinate C: Tubal tonsil D: Medial retropharyngeal lymph node E: Palatine tonsil F: Palate. (C) Lymph nodes from sheep inoculated with DGNP-DiR by nasal (IN), intradermal neck (IDn), or intradermal cheek (IDc) route.

FIGURE 3

In vitro immunostimulatory properties of the DGNP/TE vaccine in non-immunized sheep splenocytes. IL-12 and IFN-γ secreted by (A) splenocytes and (B) APC enrichment cells from four sheep. Statistical analysis was performed thanks to Kruskall–Wallis test *p < 0.05, **p < 0.01 (C) Flow cytometry characterization of splenocytes and APC enriched cells from four sheep. Statistical analysis was performed using paired t-test *p < 0.05, **p < 0.01. Data are expressed as mean ± SEM.

FIGURE 4

Humoral immune response of sheep following vaccination with DGNP/TE and infection with T. gondii. (A) Sheep from the latent experiment CTL: inoculated with DGNP alone as control; IN: vaccinated by nasal route; IDn: vaccinated by intradermal route in the neck area; and IDc: vaccinated by intradermal route in the cheek area. (B) Sheep from the transplacental transmission experiment CTL: inoculated with DGNP alone as control; IN: vaccinated by nasal route; IDn: vaccinated by intradermal route in the neck area; INF: experimentally primary infected as protected control. Specific anti-T. gondii IgG titer were explored by endpoint ELISA at D80 after the first inoculation: (A, B) n = 12/group (open symbols) and after infection (closed symbols) at the end of experiments (A) n = 10/group (B) CTL n = 9; IN n = 9; IDn n = 10; and INF n = 8 infected pregnant sheep. Median and individual data points are shown. Since axis is logarithmic, negative values are not plotted and are <25. Statistical analysis was performed thanks to two-way ANOVA (A) p < 0.0001 and (B) p = 0.0150 and Tukey’s multiple comparisons posttests *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 5

Cellular immune response in draining lymph nodes of sheep (2 per group) 6 months after the last vaccination in the latent toxoplasmosis experiment. Data are expressed as mean ± SEM. (A) Expression of MHCII + cells among draining lymph nodes cells from intranasally immunized ewes (IN) compared to CTL. (B) Expression of MHCII + cells among draining lymph nodes cells from ewes vaccinated by intradermal route in the neck area (IDn) compared to CTL. (C) Expression of MHCII + cells among draining lymph nodes cells from ewes vaccinated by intradermal route in the cheek area (IDc) compared to CTL. (D) Specific IFN-γ secretion in draining lymph nodes from ewes vaccinated by both intradermal routes compared to CTL.

FIGURE 6

Expression of immune markers of splenocytes from sheep (2 per group) 6 months after the last vaccination in the latent toxoplasmosis experiment. Splenocytes were isolated and stained for the immune markers MHCII, CD4, CD14, DU204, CD11b, CD8, and CD11c. CTL: inoculated with DGNP alone as control; IN: vaccinated by nasal route; IDn: vaccinated by intradermal route in the neck area; and IDc: vaccinated by intradermal route in the cheek area. Data are expressed as mean ± SEM.

FIGURE 7

Cellular immune response from spleen of two sheep 6 months after the last vaccination in the latent toxoplasmosis experiment. CTL: inoculated with DGNP alone as control; IN: vaccinated by nasal route; IDn: vaccinated by intradermal route in the neck area; and IDc: vaccinated by intradermal route in the cheek area. (A) Proliferation index of spleen cells measured by colorimetric BrdU. (B) IFN-γ secretion by spleen cells. (C) IL-12 secretion by spleen cells. (D) IL-10 secretion by spleen cells. Data are expressed as mean ± SEM. Statistical analysis was performed using Kruskall–Wallis test and was non-significant.

FIGURE 8

Brain cysts numeration from (A) latent toxoplasmosis experiment and (B) transplacental transmission experiment. Brain cysts load were calculated related to their individual total volume. Data are expressed as plot and mean ± SEM. Statistical analysis was performed using Kruskall–Wallis test and was non-significant. (C) Representative picture of cyst observed with an optical microscope at an original magnification of x400.