A Modified Variant of Fasciola hepatica FhSAP-2 (mFhSAP-2) as a Recombinant Vaccine Candidate Induces High-Avidity IgG2c Antibodies and Enhances T Cell Activation in C57BL/6 Mice

Abstract

Background/objectives: In the past, FhSAP-2, an 11.5 kDa recombinant protein belonging to the Fasciola hepatica saposin-like/NK-lysin family, has been shown to induce over 60% partial protection in immunized rabbits and mice when challenged with F. hepatica metacercariae. However, despite FhSAP-2 being a promising vaccine candidate, its hydrophobic nature has made its purification a challenging process. The present study aimed to determine whether a modified 9.8 kDa variant of protein (mFhSAP-2), lacking a string of 16 hydrophobic amino acids at the amino terminus and a dominant Th1 epitope, could retain its immunogenic and Th1-inducing properties.

Methods: RAW264.7 cells were stimulated with mFhSAP-2, and TNFα levels were determined. C57BL/6 mice were immunized with mFhSAP-2 alone or emulsified with Montanide ISA50. Total anti-mFhSAP-2 IgG subtypes, along with their avidity and titers, were measured using ELISA. The T cell proliferation index and levels of CD4+/CD8+ and IFNγ/IL-4 ratios were determined.

Results: In vitro, mFhSAP-2 induced dose-dependent TNFα production in RAW264.7 cells. In vivo, mice immunized with mFhSAP-2 or mFhSAP-2+ISA50 developed high-avidity IgG2a and IgG2c antibodies at levels that were significantly higher than IgG1 antibody levels. However, the mFhSAP-2+ISA50 formulation induced higher and more homogenous antibody titers than mFhSAP-2, suggesting that an adjuvant may be required to enhance mFhSAP-2 immunogenicity. Immunization with mFhSAP-2+ISA50 also induced significantly higher activated CD4+/CD8+ T cell ratios and IFNγ/IL-4 ratios compared to naïve mice.

Conclusions: Our results demonstrate that mFhSAP-2 retained its immunogenicity and Th1-polarizing properties, which were enhanced by the Montanide ISA50 adjuvant. The present study highlights the feasibility of inducing Th1-associated immune responses in mice using mFhSAP-2 as an antigen. Further studies are required to assess the potential application of the mFhSAP-2+ISA50 formulation as a vaccine against F. hepatica in natural hosts such as cattle and sheep, which could contribute to improved control and aid in the prevention and eradication of F. hepatica infection.

Keywords: C57BL6; FhSAP-2; IFNγ; IgG2a; IgG2c; avidity.

Figure 1

Strategy for cloning a modified version of recombinant FhSAP-2. (A) Sequence of 101 amino acids of FhSAP-2 (11.5 kDa) as reported in GenBank (Accession No. AF286903). The box over the first 16 amino acids represents the removed amino terminal region in mFhSAP-2, containing hydrophobic (blue) and hydrophilic (red) residues, along with a predicted signal peptide cleavage site between Ala¹⁵ and Ser¹⁶ (arrow). The amino acid sequence ¹¹AVTFA¹⁵ (underlined) represents one of the two dominant Th1 epitopes of FhSAP-2. (B) The mFhSAP-2 fusion protein sequence lacks the initial 16 amino acids and the dominant Th1 epitope. A 6His tag and a GST tag (red) were added at the amino terminus to facilitate detection and purification, respectively, and Kpnl and Hind-III restriction sites (brown) were added to mFhSAP-2 for cloning into the pET30a (+) vector. A TEV protease cleavage site (green) was also added at the amino terminus to allow the removal of the His-GST tag after purification.

Figure 2

Flowchart showing a simplified sketch of the process for the expression and purification of mFhSAP-2. IBs: Inclusion bodies. Arrows in the figure indicate the order in which each step in the process is accomplished.

Figure 3

Circular dichroism (CD) spectra of purified mFhSAP-2. The red line represents the CD spectrum, which was recorded using a Jasco J-1500 spectropolarimeter at a concentration of 200 μg/mL at 25 °C, over a wavelength range of 190 and 250 nm. CD raw data were analyzed using the BeStSel^TM web server.

Figure 4

TNFα secretion and cell viability in RAW 264.7 cells treated with mFhSAP-2. RAW264.7 cells are macrophage-like cells of murine origin. Cells were seeded in an RPMI medium at a concentration of 5 × 10⁵ cells/mL and stimulated with increasing concentrations of mFhSAP-2 (5 to 20 μg/mL). Cells stimulated with LPS (10 μg/mL) were used as an activation control, and cells treated with PBS were used as a negative control. (A) Orange line represent the OD 450nm values for each TNFα concentration of the standard curve. Dashed line shows the lineal correlation between known TNFα concentrations (x-axis) and OD₄₅₀ values (y-axis). (B) Dose–response curve (showed in blue color) showing TNFα secretion in response to mFhSAP-2 in RAW 264.7 cells. TNFα secretion in RAW 264.7 cells treated with mFhSAP-2 (20 μg/mL), LPS (10 μg/mL), and PBS (negative control). (C) Cell viability assay in RAW 264.7 cells treated with PBS, mFhSAP-2, or LPS (10 μg/mL). OD values equal to or above PBS (negative control) indicate high cell viability and metabolic activity. Data are presented as mean ± SEM. Statistical significance was determined using unpaired t-tests: **** p < 0.0001 and ns: not significant.

Figure 5

Specific IgG antibodies against mFhSAP-2 in NZW rabbits and C57BL/6 mice. The ability of mFhSAP-2 to react with sera from infected and immunized animals was measured using ELISA. (A) Levels of specific anti-mFhSAP-2 IgG antibodies elicited in New Zealand White (NZW) rabbits during early and late stages of Fasciola hepatica challenge infection. Rabbit sera collected prior to infection were used as the negative control (NC). (B) Levels of specific anti-mFhSAP-2 IgG antibodies elicited in C57BL/6 mice infected with F. hepatica or immunized either with mFhSAP-2+ISA50 or with m-FhSAP-2 alone. Sera from naïve C57BL/6 mice were used as negative controls (NC). Data are presented as mean ± SEM. Statistical significance was determined using unpaired t-tests: **** p < 0.0001, *** p = 0.0003, ** p = 0.0026, and ns: not significant.

Figure 6

IgG subtype profile induced by mFhSAP-2 in C57BL/6 mice compared to F. hepatica infection. ELISA was used to assess the IgG profile against mFhSAP-2 in C57BL/6 mice immunized with mFhSAP-2 or mFhSAP-2+ISA50 or infected with F. hepatica. (A) mFhSAP-2+ISA50 induced significantly higher IgG2c levels than IgG2a (**** p < 0.0001), IgG1 (** p = 0.0032), or IgG3 (**** p < 0.0001). (B) mFhSAP-2 also induced higher IgG2c levels than other subtypes, but the differences were not statistically significant (ns). (C) F. hepatica infection induced significantly higher IgG1 levels than IgG2a (** p = 0.0034), IgG2c (** p = 0.0040), or IgG3 (** p = 0.0016). Dashed lines in (A–C) indicate the cut-off values (OD > 0.1) for all IgG isotypes established by the average OD₄₉₂ $\pm$ 3 SD of the negative control animals. (D) F. hepatica-infected mice had significantly higher IgG1/IgG2a (*** p = 0.0002) and IgG1/IgG2c (** p = 0.0053) ratios than those immunized with mFhSAP-2+ISA50, whereas differences with those immunized with mFhSAP-2 alone were not significant (ns). Data are presented as mean ± SEM. Statistical significance was determined using unpaired t-tests. Dotter line in the figures (A–C) represent the positive cut-off value for each antibody isotype.

Figure 7

IgG2a and IgG2c avidity index and titration curves. The avidity and titers of anti-mFhSAP-2 IgG2a and IgG2c antibodies were measured using ELISA. (A) Avidity index of IgG2a and IgG2c antibodies in C57BL/6 mice either infected with F. hepatica or immunized with mFhSAP-2+ISA50. Avidity was interpreted as low if AI < 30%, intermediate if between 30% and 50%, and high if >50%. Immunization with mFhSAP-2+ISA50 induced high-avidity IgG2a and IgG2c antibodies that were significantly higher than those induced by F. hepatica infection (*** p = 0.001), which elicited moderate-to-low-avidity IgG2a and IgG2c antibodies, respectively. (B,C) The mean serum antibody titers showed that both mFhSAP-2- and mFhSAP-2+ISA50-immunized mice had lower IgG2a end-point titers (1:1600 and 1:6400, respectively) compared to IgG2c (1:3200 and 1:25,600, respectively). Data are presented as mean ± SEM.

Figure 8

Activation of CD4+ T cells in response to mFhSAP-2. (A) Splenocytes from C57BL/6 mice immunized with mFhSAP-2+ISA50 or naïve (negative control: NC) were cultured in vitro, either non-stimulated or stimulated with mFhSAP-2 for 72 h. The cell proliferation index (CPI) was calculated as the ratio of the number of cells at 72 h to the number of cells at 0 h. Non-stimulated cells from immunized mice showed a significantly higher proliferation index than those from negative controls (* p = 0.0176). (B,C) Splenocytes from C57BL/6 mice immunized with mFhSAP-2+ISA50 or naïve (negative control: NC) were cultured in vitro, stimulated with mFhSAP-2 for 24 h, and analyzed using flow cytometry. Mice immunized with mFhSAP-2+ISA50 had a significantly higher number of activated (CD69+) CD4+ T cells (* p = 0.0252) than activated CD8+ T cells compared to naive animals. CD4+ T cells from mFhSAP-2+ISA50-immunized mice showed significantly higher IFNγ/IL-4 ratios than NC animals (* p = 0.0376). Data are presented as mean ± SEM. Statistical significance was determined using unpaired t-tests.