Protection studies of an excretory-secretory protein HcABHD against Haemonchus contortus infection

Abstract

Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/β-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory-secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus.

Keywords: H. contortus; excretory–secretory protein; goats; immunization; Α/β-hydrolase.

Figure 1

Experimental protocol scheme. Goats (n = 5 for each group) were vaccinated/immunized (blue up-pointing triangle), challenged (green square), and bled (red filled circle) at various timepoints during the course of the trials. Fecal samples were collected at the indicated timepoints. A Goat experiment timeline of Trial 1 (active vaccination). B Goat experiment timeline of Trial 2 (passive immunization).

Figure 2

Parasitological determination of challenged goats in both trials. A Dynamic range of fecal egg counts (FEC) values of challenged goats in Trial 1. Goats in challenged vaccinated group (Group B) and challenged adjuvant group (Group C) started to excrete H. contortus eggs in fecal samples around Day 43, and FEC were then performed every 2 days until termination using the modified McMaster method. Eggs per gram (EPG) values were expressed as mean ± SD. Each data point represented the mean FEC value for each group (n = 5). B Determination of cumulative FEC for challenged goats in Trial 1. Cumulative FEC values were evaluated by calculating the area under the curve using the linear trapezoidal method. Cumulative FEC values (n = 5 for each group) were presented as minimum to maximum (all points). P-values were determined by non-parametric Mann–Whitney tests. C Worm burdens of each challenged group in Trial 1. Abomasum worm burdens were differentiated into male, female and total worms. The mean worm burdens (n = 5 for each group) were represented as minimum to maximum (all points), and two groups differed significantly when P < 0.05. ns not significant. D Dynamic range of FEC values of challenged goats in Trial 2. Goats in challenged immunized group (Group E) and challenged control group (Group F) began to excrete H. contortus eggs in feces around Day 29, and FEC were then performed every other day until the end of the trial. EPG values were represented as mean ± SD. Each data point represented the mean FEC value for each group (n = 5). E Determination of cumulative FEC for challenged goats in Trial 2. Cumulative FEC values were determined by calculating the area under the curve as well. Cumulative FEC values (n = 5 for each group) were presented as minimum to maximum (all points). P-values were determined by non-parametric Mann–Whitney tests. F Worm burdens of challenged goats in Trial 2. The mean worm burdens (n = 5 for each group) were denoted as minimum to maximum (all points), and two groups differed significantly when P < 0.05. ns not significant.

Figure 3

Determination of abomasal mucosal antibody responses. Abomasal swab samples were collected at post-mortem from goats in unchallenged adjuvant group (Group A), challenged vaccinated group (Group B) and challenged adjuvant group (Group C), unchallenged control group (Group D), challenged immunized group (Group E) and challenged control group (Group F), and subjected to mucosal IgA, IgE and IgG determinations via ELISA assays. A Determination of mucosal IgA levels in Trial 1. B Determination of mucosal IgA levels in Trial 2. C Determination of mucosal IgE levels in Trial 1. D Determination of mucosal IgE levels in Trial 2. E Determination of mucosal IgG levels in Trial 1. F Determination of mucosal IgG levels in Trial 2. Mucosal antibody levels for each group (n = 5) were represented as minimum to maximum (all points) and non-parametric Kruskal–Wallis multiple comparisons were performed for statistical analysis (P < 0.05).

Figure 4

Dynamics of serum anti-rHcABHD IgG and serum total IgG levels in active and passive immunization trials. Blood samples were obtained at various timepoints from goats in the unchallenged adjuvant group (Group A), challenged vaccinated group (Group B) and challenged adjuvant group (Group C), unchallenged control group (Group D), challenged immunized group (Group E) and challenged control group (Group F), and serum samples were harvested and assayed for the determination of serum anti-rHcABHD IgG and total IgG levels. A Dynamics of serum anti-rHcABHD IgG levels in Trial 1. B Dynamics of serum anti-rHcABHD IgG levels in Trial 2. C Dynamics of serum total IgG levels in Trial 1. D Dynamics of serum total IgG levels in Trial 2. Each data point denoted the mean serum antibody levels (mean ± SD, n = 5 for each group) and asterisks represented statistically significant differences compared to Group C in Trial 1 (*P < 0.05 and ****P < 0.0001) and compared to Group F in Trial 2 (*P < 0.05 and ****P < 0.0001), respectively.

Figure 5

Blood pathology determination of eosinophil numbers, hemoglobin levels and hematocrit values in active and passive immunization trials. Fresh blood samples were obtained at each sampling day from goats in unchallenged adjuvant group (Group A), challenged vaccinated group (Group B) and challenged adjuvant group (Group C), unchallenged control group (Group D), challenged immunized group (Group E) and challenged control group (Group F), and subjected to complete blood count (CBC) determination to monitor their health conditions. A Dynamics of the numbers of blood eosinophils in Trial 1. B Dynamics of the numbers of blood eosinophils in Trial 2. C Dynamics of hemoglobin levels in Trial 1. D Dynamics of hemoglobin levels in Trial 2. E Dynamics of hematocrit values in Trial 1. F Dynamics of hematocrit values in Trial 2. Eosinophil numbers, hemoglobin levels and hematocrit values (n = 5 for each group) were represented as mean ± SD, and asterisks represented statistically significant differences compared to Group A in Trial 1 (*P < 0.05 and **P < 0.01) and compared to Group D in Trial 2 (*P < 0.05 and **P < 0.01), respectively.

Figure 6

Circulating cytokine production profiles in unchallenged and challenged goats throughout the trials. Serum samples obtained at different timepoints from goats in unchallenged adjuvant group (Group A), challenged vaccinated group (Group B), challenged adjuvant group (Group C), unchallenged control group (Group D), challenged immunized group (Group E) and challenged control group (Group F) were subjected to the determination of cytokine production levels. A Dynamics of circulating IL-2 levels in Trial 1. B Dynamics of circulating IL-2 levels in Trial 2. C Dynamics of circulating IL-4 levels in Trial 1. D Dynamics of circulating IL-4 levels in Trial 2. E Dynamics of circulating IL-17A levels in Trial 1. F Dynamics of circulating IL-17A levels in Trial 2. The mean cytokine production levels (n = 5 for each group) were denoted as mean ± SD, and different letters represented significant differences between groups (P < 0.05) at the sampling day.