Adjuvant-enhanced antibody and cellular responses to inclusion bodies expressing FhSAP2 correlates with protection of mice to Fasciola hepatica

Abstract

Fasciola hepatica saposin-like protein-2 (FhSAP2) is a protein differentially expressed in various developmental stages of F. hepatica. Recombinant FhSAP2 has demonstrated the induction of partial protection in mice and rabbits when it is administered subcutaneously (SC) in Freund's adjuvant. Because FhSAP2 is overexpressed in bacteria in the form of inclusion bodies (IBs), we isolated IBs expressing FhSAP2 and tested their immunogenicity when administered SC in mice emulsified in two different adjuvants: QS-21 and Montanide TM ISA720. Animals received three injections containing 20 μg of protein two weeks apart and 4 weeks after the third injection, mice were infected with 10 F. hepatica metacercariae by oral route. The percentages of protection induced by FhSAP2-IBs were estimated to be between 60.0 and 62.5% when compared with adjuvant-vaccinated, infected controls. By determining the levels of IgG1 and IgG2a antibodies and IL-4 and IFNγ cytokines in the serum of experimental animals, it was found that both Th1 and Th2 immune responses were significantly increased in the FhSAP2-IBs vaccinated groups compared with the adjuvant-vaccinated, infected control groups. The adjuvant-vaccinated groups had significantly lower IgG1 to IgG2a ratios and lower IL-4 to IFNγ ratios than the FhSAP2-IBs vaccinated animals, which is indicative of higher levels of Th2 immune responses. Irrespective to the adjuvant used, animals vaccinated with FhSAP2-IBs exhibited significantly higher survival percentage and less liver damage than the adjuvant-control groups. This study suggests that FhSAP2 has potential as vaccine against F. hepatica and that the protection elicited by this molecule could be linked to a mechanism driven by the CD4-Th1 cells.

Keywords: Fasciola hepatica; Inclusion bodies; Saposin-like proteins; Vaccination.

Figure-1. Western blot analysis of inclusion bodies containing FhSAP2

After expression inclusion bodies were isolated and analyzed by 15% SDS-PAGE. Proteins were electrotransferred to a nitrocellulose (NC) sheet and after blocking were probed with anti-Xpress™ epitope-peroxidase labeled antibody. A strong major band of ~16.4kDa was revealed, which represent the predicted 11.5kDa polypeptide band of FhSAP2 plus 4.9kDa that include the 6x (His)-tag plus the Xpress epitope at the amino terminus of the protein moiety. In addition, a very weak protein band of ~31kDa was also observed, which could represent dimeric forms of FhSAP2.

Figure-2. Survival analysis

Kapplan-Meier analysis to estimate the survival probability over the time after challenge in mice immunized with FhSAP2-IBs in QS-21 or Montanide™ ISA720 compared to mice vaccinated with E. coli proteins in QS-21 or Montanide™ ISA720 adjuvants. Log-rank test determined that there are statistical differences (p <0.05) between survivals probabilities of FhSAP2-IBs vaccinated groups compared to their corresponding adjuvant-vaccinated, infected control group.

Figure-3. Antibody response to F. hepatica ES products in animals immunized with FhSAP2-IBs after the challenge infection

(A) Specific anti-ES products IgG1 and IgG2a antibody levels elicited in mice vaccinated with FhSAP2-IBs in QS-21 or Montanide™ ISA720 were measured at euthanasia, which occurred 45-days after the challenge infection. ** Indicate significant differences (p<0.0001) between the levels of IgG1 and IgG2a of each FhSAP2-IBs vaccinated or control group. Values represent the mean ± SD of triplicate ELISA readings of each experimental group. (B) ** Differences highly significant (p<0.0001) were found between the IgG1 to IgG2a ratios between FhSAP2-IBs vaccinated groups compared with their corresponding control group.

Figure-4. Activation status of spleen lymphocytes from FhSAP2-IBs immunized animals prior to the challenge infection

Spleen lymphocytes from FhSAP2-IBs immunized animals were analyzed by flow cytometry. CD69+ was used as marker of activation. Results demonstrate that both formulations significantly activate significantly higher CD4+ T-cells than CD8+ T-cells population.

Figure-5. Production of cytokines in serum from animals immunized with FhSAP2-IBs after the challenge infection

We used BioPlex for determining levels of IFNγ, IL-4 (A) and TNFα (B) cytokine in serum of animals vaccinated with FhSAP2-IBs in QS-21 or Montanide™ ISA720 that were euthanized 45-days after the challenge infection. Results are compared to those obtained in the control groups. *** QS-21 or Montanide™ ISA720-control groups had significantly more amount of IL-4 than FhSAP2-IBs vaccinated groups. The highest IL-4 to IFNγ ratios were found in the controls, which indicate polarized Th2 immune responses. The highest levels of serum TNFα were found in animals from groups vaccinated with FhSAP2-IBs in QS-21 (p=0.0016) compared to those immunized with FhSAP2-IBs in Montanide™ ISA720 or control animals.