Protective Immune Responses Generated in a Murine Model Following Immunization with Recombinant Schistosoma japonicum Insulin Receptor

Abstract

There is a pressing need to develop vaccines for schistosomiasis given the current heavy dependency on praziquantel as the only available drug for treatment. We previously showed the ligand domain of the Schistosoma japonicum insulin receptor 1 and 2 (rSjLD1 and 2) fusion proteins conferred solid protection in mice against challenge infection with S. japonicum. To improve vaccine efficacy, we compared the immunogenicity and protective efficacy of rSjLD1 on its own and in combination with S. japonicum triose-phosphate isomerase (SjTPI), formulated with either of two adjuvants (QuilA and montanide ISA 720VG) in murine vaccine trials against S. japonicum challenge. The level of protection was higher in mice vaccinated only with rSjLD1 formulated with either adjuvant; rSjTPI or the rSjTPI-rSjLD1 combination resulted in a lower level of protection. Mirroring our previous results, there were significant reductions in the number of female worms (30⁻44%), faecal eggs (61⁻68%), liver eggs (44⁻56%), intestinal eggs (46⁻48%) and mature intestinal eggs (58⁻63%) in the rSjLD1-vaccinated mice compared with the adjuvant only groups. At 6-weeks post-cercarial challenge, a significantly increased production of interferon gamma (IFNγ) in rSjLD1-stimulated splenic CD4⁺ T cells was observed in the rSjLD1-vaccinated mice suggesting a Th1-type response is associated with the generated level of protective efficacy.

Keywords: Schistosoma japonicum; insulin receptor; murine model; triose-phosphate isomerase; vaccine.

Figure 1

Kinetics of specific IgG and IgG1 and IgG2a antibody isotypes induced in mice immunised with the rSjLD1 and rSjTPI vaccine, respectively, conjugated with QuilA or ISA720. (a) Anti-rSjLD1 specific IgG, IgG1 and IgG2a antibody isotype levels (OD450) in mice vaccinated with rSjLD1 + QuilA or rSjLD1 + ISA and (b) Anti-rSjTPI specific IgG, IgG1 and IgG2a antibody isotype levels in mice vaccinated with rSjTPI + QuilA or rSjTPI + ISA are shown over the 12 weeks after the primary vaccination and include time points at week 0, weeks 2 and 4 after the primary vaccination (2 boosts), week 6 (just prior to challenge with S. japonicum cercariae) and week 12 (just prior to perfusion).

Figure 2

The cytokine profiles of splenocytes recovered from mice vaccinated with rSjLD1, rSjTPI and rSjLD1 + SjTPI adjuvanted with ISA720 at 6 weeks post-challenge. Splenocytes isolated from vaccinated and adjuvant control mice were stimulated with medium (as a control), rSjLD1, rSjTPI, SEA and SWAP for 72 h, followed by intracellular cytokine staining. After exclusion of doublets and dead cells identified with the live/dead marker, the CD3⁺ population (total T cells) were further classified into CD4⁺ and CD8⁺ subsets. Total splenocytes, CD4⁺ T cells and CD8⁺ T cells isolated from each spleen were determined (a) and the proportion of IFNγ and IL-4 producing splenic CD4⁺ or CD8⁺ T cells were determined after stimulation with (b) rSjLD1; (c) rSjTPI; (d) SWAP; and (e) SEA (* p value ≤ 0.05; ** p value ≤ 0.001; *** p value ≤ 0.0001).

Figure 3

Cytokine profiles of splenic CD4⁺ T cells recovered from mice vaccinated with SjLD1, SjTPI and SjLD1 + SjTPI adjuvanted with QuilA at 6 weeks post-challenge. Splenocytes isolated from vaccinated and adjuvant control mice were stimulated with media, rSjLD1, rSjTPI, SEA and SWAP for 72 h, followed by intracellular cytokine staining. The proportion of IFNγ- and IL-4-producing splenic CD4⁺ T cells were determined after stimulation with (a) rSjLD1; (b) rSjTPI; (c) SWAP and (d) SEA (* p value ≤ 0.05; ** p value ≤ 0.001).

Figure 4

Ratio of Th1/Th2 in splenic CD4⁺ T cells obtained from mice vaccinated with SjLD1, SjTPI and SjLD1 + SjTPI adjuvanted with ISA or QuilA at 6 weeks post-challenge. The ratio of Th1/Th2 was determined by the proportion of IFNγ- and IL-4-producing splenic CD4⁺ T cells isolated from mice vaccinated with SjLD1, SjTPI and SjLD1 + SjTPI adjuvanted with (a) ISA and (b) QuilA, respectively, after stimulation with rSjLD1, rSjTPI, SWAP and SEA (* p value ≤ 0.05; ** p value ≤ 0.001).

Figure 5

Blood glucose levels of mice were monitored while fasting (no food given overnight) and 2 h after given a meal (a) following the 3rd vaccination with rSjLD1 or adjuvant controls and (b) at 6 weeks post-challenge (before worm perfusion).

Figure 6

Immunofluorescence staining of SjTPI in hepatic stellate cells (HSC). Permeabilized HSC, after blocking with DAKO, were incubated with rabbit anti-actin antibody mixed with (a) naïve mouse serum or (b) mouse anti-rSjTPI serum. HSC from A and B were then incubated with a mixture of goat anti-mouse Alexa-488 (labeled with green fluorescence dye) and goat anti-rabbit Alexa-647 (labeled with red fluorescence dye). HSC nuclei were stained blue with DAPI. (c) TPI activity assays of supernatants and HSC lysates after culture in DMEM containing 5% (v/v) mouse anti-SjTPI serum or naïve mouse serum.