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. 2022 Nov 27;7(12):401.
doi: 10.3390/tropicalmed7120401.

Nano-Encapsulated Melatonin: A Promising Mucosal Adjuvant in Intranasal Immunization against Chronic Experimental T. gondii Infection

Doaa E Said  1 , Eglal I Amer  1 , Eman Sheta  2 , Shaimaa Makled  3 , Hala E Diab  1 , Fadwa M Arafa  1
Affiliations

Nano-Encapsulated Melatonin: A Promising Mucosal Adjuvant in Intranasal Immunization against Chronic Experimental T. gondii Infection

Doaa E Said et al. Trop Med Infect Dis. .

Abstract

Melatonin (MLT) is now emerging as one of the universally accepted immunostimulators with broad applications in medicine. It is a biological manipulator of the immune system, including mucosal ones. MLT was encapsulated in solid lipid nanoparticles (SLNs), then 100 mg/kg/dose of MLT-SLNs was used as an adjuvant of Toxoplasma lysate antigen (TLA). Experimental mice were intra-nasally inoculated with three doses of different regimens every two weeks, then challenged with 20 cysts of T. gondii Me49 strain, where they were sacrificed four weeks post-infection. Protective vaccine efficacy was evident via the significant brain cyst count reduction of 58.6%, together with remarkably high levels of humoral systemic and mucosal anti-Toxoplasma antibodies (Ig G, Ig A), supported by a reduced tachyzoites invasion of Vero cells in vitro upon incubation with sera obtained from these vaccinated mice. A cellular immune response was evident through the induction of significant levels of interferon-gamma (IFN γ), associated with morphological deteriorations of cysts harvested from the brains of vaccinated mice. Furthermore, the amelioration of infection-induced oxidative stress (OS) and histopathological changes were evident in mice immunized with TLA/MLT-SLNs. In conclusion, the present study highlighted the promising role of intranasal MLT-SLNs as a novel mucosal adjuvant candidate against chronic toxoplasmosis.

Keywords: Ig A; Toxoplasma gondii; antioxidant; immunization; intranasal; melatonin; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 2
Figure 2
Colloidal characterization of freshly prepared plain and MLT-SLNs suspensions (at zero-day). (A) Particle size curve distribution for plain SLNs. (B) Zeta potential curve of plain SLNs. (C) Particle size distribution curve of MLT-SLNs. (D) Zeta potential curve of MLT-SLNs.
Figure 3
Figure 3
TEM of plain and adjuvant loaded SLNs (×30,000). (A) Plain SLNs showing rounded smooth surfaces with sizes ranging from 83.87–106.81 nm. (B) MLT-SLNs rounded smooth surfaces with sizes ranging from 278.12–330.4 nm.
Figure 1
Figure 1
Flowchart representing the time schedule of the experimental study.
Figure 4
Figure 4
In vitro release profile of MLT from SLN dispersion versus free MLT. n = 3. Data represented as mean ± SD.
Figure 5
Figure 5
The storage stability of MLT-SLNs upon storage for six months at 4 °C using particle size analysis (A), PDI (B), zeta potential (C), and EE (D).
Figure 6
Figure 6
Parasitological study. (A) In vitro tachyzoite invasion and replication assay in Vero cells. Tachyzoites of the virulent RH strain were pre-incubated with sera from naïve mice collected on day 0, or with sera collected from mice vaccinated with different studied regimens on day 42 from the beginning of the experiment (i.e., before infection). Dots represent the mean number of T. gondii tachyzoites (×104) per well in the culture supernatant of Vero cells after incubation. In vitro assays were performed with pooled serum samples (n = 3) repeated three times. Results represent data from three independent experiments, (B) mean of T. gondii cyst count and (C) cyst size in brain of mice of different studied groups at week 4 after the in vivo infection challenge with 20 cysts of the avirulent Me49 Toxoplasma strain. Data are expressed as mean ± SD. *** p < 0.001. Group I: Control, Group II: MLT-SLNs, Group III: TLA, Group IV: TLA/ plain SLNs, Group V: TLA/ MLT-SLNs.
Figure 7
Figure 7
SEM of T. gondii cyst harvested from the infected control group (I); (A) mice vaccinated with TLA (group III); (B) mice vaccinated with TLA/MLT-SLNs (group V) (C,D). (A) Cysts harvested from the control group showed regular spherical-shaped bodies with some fine dimples and indentations (×12,000). (B) Cysts from group III showed mild roughness and surface irregularities with retained spherical outlines (×10,000). (C,D) Cysts from group V showed mild swelling and surface irregularities with a focal blebs formation (arrows) (×9500 and ×10,000, respectively).
Figure 8
Figure 8
Pre-infection and post-infection serum levels of anti-Toxoplasma IgG (OD) among different studied groups. (A) Anti-Toxoplasma IgG antibody levels in sera of mice two weeks following the last booster dose of immunization (pre-infection). (B) Anti-Toxoplasma IgG antibody level in sera of mice four weeks after challenging the infection (post-infection). Data were expressed by using Mean ± SD. ***: Statistically significant at p ≤ 0.001. *: Statistically significant at p ≤ 0.05. Group I: Control Group II: MLT–SLNs, Group III: TLA. Group IV: TLA/ MLT plain SLNs, Group V: TLA/ MLT–SLNs.
Figure 9
Figure 9
Pre-infection and post-infection intestinal levels of anti-Toxoplasma IgA (OD) among different studied groups. (A) Anti-Toxoplasma IgA antibody levels in intestinal washes of mice two weeks after completion of immunization schedules (pre-infection). (B) Anti-Toxoplasma IgA antibody level in intestinal washes of mice four weeks after oral challenge with Me49 Toxoplasma cysts (post-infection). Data were expressed by using Mean ± SD. ***: Statistically significant at p ≤ 0.001. Group I: Control, Group II: MLT–SLNs, Group III: TLA, Group IV: TLA/ MLT plain SLNs, Group V: TLA/ MLT–SLNs.
Figure 10
Figure 10
Pre-infection and post-infection splenic cell culture IFN-γ production among different studied groups. (A) INF-γ concentration (pg/mL) in the supernatant of splenic cell suspension two weeks after the last immunization dose (pre-infection). (B) INF-γ concentration (pg/mL) in the supernatant of splenic cell suspension four weeks following infection (post-infection). Data were expressed by using Mean ± SD. ***: Statistically significant at p ≤ 0.001. Group I: Control, Group II: MLT–SLNs, Group III: TLA, Group IV: TLA/ MLT plain SLNs, Group V: TLA/ MLT-SLNs.
Figure 11
Figure 11
Histopathological findings in brain sections of the infected control group (A–E), and vaccinated groups (F,G). (A) H&E-stained section showing an oval, well-defined T. gondii cyst in the cerebral cortex containing multiple bradyzoites (×400), (B) H&E-stained section showing severe inflammatory infiltration of meninges on the surface of the cerebral cortex (×100), (C) H&E-stained section showing perivascular lymphocytic inflammatory infiltrate (×100), (D) H&E-stained section showing microglial nodules (×100), (E) H&E-stained section showing increased cellularity throughout the brain parenchyma (×100), (F) H&E-stained section showing cysts with a degenerated irregular cyst wall in the cerebral cortex (×400), (G) H&E-stained section showing an improvement in inflammatory changes with neither meningeal infiltration nor cerebral-increased cellularity (×100).
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