The study focused on IPSE/α-1, an abundant glycoprotein secreted by S. mansoni eggs that participates in host–parasite interactions and is known for inducing IL-4 production and supporting a Th2-associated response. Retained eggs drive the granulomatous inflammation and fibrosis associated with intestinal schistosomiasis, making egg-secreted proteins biologically relevant targets for vaccine research. The investigators selected IPSE/α-1 to examine its immunomodulatory potential after DNA delivery, rather than because it had demonstrated superiority to other schistosomiasis vaccine candidates such as Sm14. Its intrinsic ability to alter cytokine production also means that a systemic response after vaccination cannot, by itself, be interpreted as evidence of protective immunity.

For construct preparation, the researchers removed rare codons and selected mammalian-preferred codons to improve the likelihood of efficient translation, while introducing HindIII and KpnI restriction sites for directional cloning. The optimized sequence was synthesized, inserted into pVAX-1 with T4 DNA ligase and transformed into chemically competent Escherichia coli DH5α cells. Five colonies were screened by colony PCR, and positive colonies were cultured for plasmid extraction. The expected PCR product supported successful insertion, although the final construct and its cloning junctions were not independently verified by Sanger sequencing, and transgene expression in mammalian cells was not measured directly.

The plasmid was then complexed with liposomes through thin-film hydration and manual extrusion. DC-Chol served as the cationic lipid and DOPE as the helper lipid, both mixed at a 1:1 molar ratio. Six nitrogen-to-phosphate charge ratios, from 1:1 through 6:1, were prepared by varying the ratio of positively charged amine groups to negatively charged DNA phosphate groups. After the lipids were dissolved in chloroform and methanol, the organic phase was evaporated under nitrogen and the resulting film was hydrated with the DNA solution. The mixture was extruded 21 times through a 200-nanometer polycarbonate membrane at 50 °C to improve vesicle uniformity. Dynamic light scattering, zeta-potential measurement and agarose gel electrophoresis were performed in triplicate.

The optimized 3:1 formulation had a relatively homogeneous size distribution below 200 nm and a positive zeta potential of approximately +30 mV, consistent with electrostatic association between the cationic liposomes and the plasmid. In the gel-retardation assay, residual migrating DNA was visible at 1:1 and 2:1, whereas no clear free-DNA band was detected at 3:1 or higher under the assay conditions. Densitometry estimated that about 91.9% of the signal was retained or retarded in the 1:1 formulation, while higher ratios showed no clear bands in the naked-DNA migration region. These observations support efficient association between plasmid and liposome, but the investigators did not independently measure absolute encapsulation efficiency by separating free from particle-associated DNA.

Cell compatibility was tested after 24 hours in human foreskin fibroblast HFF-1 cells exposed to formulation concentrations from 0.125 to 16 μg/mL. The MTS assay indicated 109.82% ± 2.14 metabolic activity at 0.125 μg/mL and more than 85% viability at 0.25 and 0.5 μg/mL. Viability declined with increasing dose: it was 66.20% at 2 μg/mL, 35.06% at 4 μg/mL, 19.79% at 8 μg/mL and 13.92% at 16 μg/mL. The estimated median cytotoxic concentration was therefore between 2 and 4 μg/mL under the tested conditions. The authors characterized the formulation as having low cytotoxicity at concentrations of 1 μg/mL or less, but the cell assay does not establish its safety in animals or humans.

For the animal experiment, 100 healthy male Swiss albino mice aged 14 weeks and weighing 25–30 g were randomly assigned to phosphate-buffered saline or to 1, 2 or 3 μg/mL of the formulated candidate. Each group contained 25 mice, with five sacrificed at 24 hours, 48 hours, seven days, 14 days or 21 days after administration, producing five animals per dose and time point. Serum IFN-γ, IL-4 and IL-10 were measured with a multiplex bead assay. IFN-γ was lower in all vaccinated groups during the first 24–48 hours. It remained reduced in the 1 and 3 μg/mL groups on day seven, while the 2 μg/mL group showed partial recovery. Levels rose markedly on day 14, particularly at 1 and 2 μg/mL, and the 2 μg/mL group had the highest value on day 21. These systemic measurements do not establish antigen-specific cellular immunity or Th1 polarization.

IL-4 showed a different temporal profile. There were no significant differences among groups at 24 hours, but concentrations increased markedly by 48 hours, especially in the 2 μg/mL group. The 2 μg/mL dose continued to produce the highest IL-4 concentrations on days seven, 14 and 21, while the 3 μg/mL dose did not generate a larger response. The 1 μg/mL group showed a more moderate rise at 48 hours, a peak around day seven and a subsequent decline. Because the cellular sources and antigen specificity of IL-4 were not determined, the observed pattern cannot establish antigen-specific Th2 polarization. It also cannot be extended directly to the antibody responses associated with resistance to human reinfection, because no IPSE/α-1-specific IgG, IgG subclass or IgE measurements were performed.

IL-10, a cytokine associated with regulation of inflammatory and lymphocyte responses, rose rapidly within 24 hours, particularly at 2 and 3 μg/mL, and reached its highest early values at 3 μg/mL on day two. Levels declined across the groups by day seven, when differences were no longer significant. IL-10 increased again on day 14, with the 2 μg/mL group peaking, and remained elevated in that group on day 21; the 3 μg/mL group showed a moderate late increase that did not match its early peak. The simultaneous movement of IFN-γ, IL-4 and IL-10 indicates a mixed systemic cytokine response involving pro-inflammatory, Th2-associated and regulatory signals. It does not demonstrate the participation of particular regulatory T-cell populations, because the investigators did not phenotypically characterize immune cells or measure antigen-specific responses.

Overall, the experiments provide a formulation and immunomodulation dataset for a liposome-complexed IPSE/α-1 DNA candidate, not evidence that a vaccine is effective. The formulation combined nanoscale dimensions, positive surface charge, strong gel retention of plasmid and limited short-term cytotoxicity at lower concentrations, while the mouse experiment identified a distinct cytokine trajectory associated with dose and time. Important gaps remain: the final DNA sequence was not confirmed by sequencing, mammalian expression was not established, cellular uptake and transfection were not directly assessed, and neither antibody responses nor protective efficacy against S. mansoni challenge were tested. The investigators therefore identify antigen-specific humoral and cellular assays, expression testing, quantitative encapsulation measurements and infection-challenge studies as necessary next steps before drawing conclusions about immunogenicity, safety or protection against schistosomiasis.

Subject of Research: Preclinical formulation and cytokine assessment of a liposomal DNA vaccine targeting the Schistosoma mansoni egg antigen IPSE/α-1.

Article Title: Development, characterization, and immunomodulatory evaluation of a liposomal DNA vaccine targeting Schistosoma mansoni IPSE/α-1

Article References: Alturbak, D. A., Alshehri, A. A., Almughem, F. A., Jarallah, S. J., Alouffi, A., Marie, M. A., Elnagar, D. M., & Aljasham, A. T. (2026). Development, characterization, and immunomodulatory evaluation of a liposomal DNA vaccine targeting Schistosoma mansoni IPSE/α-1. Frontiers in Veterinary Science, 13, Article 1910156. https://doi.org/10.3389/fvets.2026.1910156

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1910156

Keywords: Schistosoma mansoni, schistosomiasis, DNA vaccine, IPSE/α-1, liposomes, DC-Chol, nanoparticles, cytokine modulation, mouse study, parasite immunology, Development, characterization