In silico prediction of CD4+ T cell and B cell epitopes from the peptidase and GTPase protein families of Haemonchus contortus, and their antigenicity in vitro

Abstract

Haemonchus contortus is a highly pathogenic parasitic nematode that infects small ruminants such as sheep and goats, predominantly in subtropical and tropical regions. To support the development of alternative vaccines against H. contortus, this study applied an immunoinformatic approach to identify potential CD4+ T cell and B cell epitopes from peptidase and GTPase proteins and further tested their antigenicity in vitro. Several immunoinformatic tools predicted conserved epitopes based on antigenicity, allergenicity, and toxicity. High-scoring antigenic, non-allergenic, and non-toxic peptides were chemically synthesized for testing. Six naïve goats were orally infected and challenged with H. contortus third-stage larvae. Immune abomasal lymph nodes were collected on day 71, and immune sera were collected on days 21 and 63 post-infection for in vitro assays measuring T cell activation through lymphocyte proliferation assays and humoral responses via enzyme-linked immunosorbent assays (ELISA). Ten peptides were predicted. These included eight CD4+ T cell peptides predicted to induce IL-4 only and two conserved linear B cell peptides. They were identified from three proteins: (1) Peptidase M12A and Metridin ShK toxin domain-containing protein (Hc-PP-1, Hc-PP-5, Hc-PP-6, Hc-PP-7, Hc-PP-8), (2) Peptidase M1 domain-containing protein (Hc-PP-2, Hc-PP-9, Hc-PP-9-B1), and (3) RhoGAP domain-containing protein (Hc-PP-4, Hc-PP-4-B1). In vitro tests showed that four peptides (Hc-PP-2, Hc-PP-4, Hc-PP-5, Hc-PP-9) exhibited high stimulation indexes but were not significantly different from controls and were regarded as negative for inducing lymphocyte proliferation. Only Hc-PP-4-B1 stimulated significant proliferation (p ≤ 0.030), suggesting strong T cell activation. However, when Hc-PP-4-B1 was combined with its corresponding CD4+ T cell peptide Hc-PP-4, proliferation was inhibited, indicating a negative interaction. ELISA tests with immune sera on day 21 showed that Hc-PP-4 (p ≤ 0.051) and Hc-PP-5 (p ≤ 0.058) were significantly recognised by IgG antibodies. Other peptides and combinations showed high but non-significant responses. By day 63, multiple peptides and combinations maintained high IgG levels without significant differences, with overall antibody recognition increasing post-secondary infection. Hc-PP-4 and Hc-PP-4-B1 showed stronger IgG responses after primary infection and diminished performance when combined, supporting their effectiveness as individual peptides. Peptide Hc-PP-8 neither induced proliferation nor was recognised by IgG. In conclusion, out of the ten predicted epitopes, three (Hc-PP-4-B1, Hc-PP-4, and Hc-PP-5) demonstrated antigenicity in vitro by inducing lymphocyte proliferation and being recognised by immune sera. Some peptide combinations, particularly Hc-PP-4 with Hc-PP-4-B1, inhibited immune responses. These findings reveal that in silico predictions in this study did not correlate with in vitro outcomes, underscoring the need for experimental validation due to limitations in the predictive accuracy of computational tools and the complex nature of immune responses. Nonetheless, this study provides a basis for further research into the validated epitopes as potential novel vaccine candidates to control H. contortus infection and address the growing threat of anthelmintic resistance in small ruminants.