Antigens and polymers
Whole adult male and female live H. contortus parasites were obtained opportunistically from naturally infected animals in Iowa, USA and initially frozen in PBS. A whole-killed vaccine was prepared by homogenizing adult worms in a pyrex tissue grinder with cold PBS. The protein concentration of the homogenate was determined by bicinchoninic acid assay and stored at − 20 °C in 1 mL aliquots. This concentration was used to normalize the antigen load throughout the vaccine compounding process which included both soluble and insoluble total whole-killed antigen and referred to as total H. contortus antigen.
Polyanhydride (PyAn) copolymers were based on a molar composition of 20% 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 80% 1,6-bis(p-carboxyphenoxy)hexane (CPH) (20:80 CPTEG: CPH). 20:80 CPTEG: CPH copolymers were synthesized using melt polycondensation as described previously16. The number-average molecular weight and molar composition was quantified using1H NMR (DXR 500), specifically by end group analysis. The molecular weights and molar compositions of the various batches of 20:80 CPTEG: CPH copolymers were consistent with previous reports, with molecular weights between 5000 and 10,000 Daltons and molar composition within 5% of the desired 20:80 molar ratio.
Vaccine formulations
The soluble vaccine antigen/adjuvant formulation was made by emulsifying 300 µg of total H. contortus antigen in 0.5 mL phosphate buffered saline (PBS) with 0.5 mL Montanide ISA 61 VG Water and Oil adjuvant (Seppic) designed for long-term immunity with a total soluble vaccine volume of 1 mL/dose. The group receiving only the soluble antigen/adjuvant vaccine (soluble group) were given 900 μg of H. contortus antigen emulsified in 1 mL total volume of Montanide and PBS. This controlled for total antigen load, since the other experimental groups also received 900 μg antigen but distributed into soluble, PyAn, or VPEAR systems described below.
The PyAn (free) rods were formed and pressed as described previously27,28. Each rod consisted of 210 mg PyAn, 300 µg parasite antigen, and 75 mg of DEAE-dextran adjuvant. Parasite antigen, PyAn and adjuvant were solubilized in 1 mL methylene chloride, mixed, air-dried and then pressed into rods under 0.5 tons-on-ram for 5 s.
The VPEAR polyethylene implant was similarly loaded with 140 mg PyAn, 300 µg parasite antigen, and adjuvant. One experimental group adjuvant had 25 mg of DEAE-dextran (DD) while the other experimental group had 500 µg Quil-A, (DQ). Loaded PyAn was placed into the polyethylene implant and overlayed with 100 µl 5% bovine collagen (EZ-gel, Advanced BioMatrix) and cross-linked as described previously27,28. The 0.45 μm porous PVDF membrane cap was adhered with n-butyl cyanoacrylate and stored at 4 °C in PBS prior to administration.
The adjuvant-only group consisted of the soluble vaccine of 0.5 mL PBS emulsified with 0.5 mL Montanide adjuvant, a PyAn (free) rod with DEAE-dextran adjuvant and no parasite antigen, and a VPEAR with DEAE-dextran adjuvant and no parasite antigen. To maximize the numbers of animals per treatment group only the DEAE-dextran (DD) control was chosen for comparison given that it contained the highest amount of adjuvant by weight.
Experimental groups
A summary of animal groups is shown in Table 1. The four experimental groups were (1) “soluble” (900 µg of H. contortus antigen in Montanide ISA 61), (2) “DD” (combination of 300 µg soluble antigen in montanide, a free rod with 300 µg antigen with DEAE-dextran, and a VPEAR implant with 300 µg antigen and with DEAE–dextran), (3) “DQ” combination of 300 µg soluble antigen in montanide, PyAn with 300 µg antigen with DEAE-dextran, and VPEAR with 300 µg antigen with quil A, and (4) “adjuvant only” (same as group 2, but with no H. contortus antigens in any of the 3 vaccine components). In summary, 3 groups all received 900 µg of antigen but distributed in different forms while 1 group received adjuvant only.
Animals
All experiments were conducted in accordance with applicable laws, were in accordance with ARRIVE guidelines, and were approved by the university institutional animal care and use committee under protocol 19–109. Mixed breed white-faced lambs (25–30 kg) were randomly assigned to one of the four treatment groups (n = 6/group). Lambs were sourced from the university sheep teaching farm. Lambs were born in indoor sheds with a dirt floor, and were never allowed to be on pasture. Sheep were maintained under trichostrongyle-free conditions for the entire study which was confirmed by centrifugal fecal flotation at the time of purchase, prior to vaccination, and prior to challenge.
Soluble vaccine was administered in the neck via 18-gauge needle into subcutaneous tissue. Local lidocaine anesthesia was administered and a stab incision facilitated the insertion of a commercial implant needle (Compudose, Elanco) into the neck which was used to administer the free rod and VPEAR. A single interrupted suture was placed to ensure tissue healing. Jugular venipuncture was used to obtain blood for monitoring serum antibodies during the duration of the study. Sheep were vaccinated during a single event at 3 months of age and then maintained under trichostrongyle-free conditions for the entire study period. Animals were not shedding trichostrongyle eggs as determined by centrifugal fecal flotation until the time of challenge with a field isolate of H. contortus.
47 weeks post-vaccination, sheep were challenged with H. contortus. Larva from the same source were stored at 4 °C in PBS and the number of live larvae were determined by microscopy. Animals were challenged with approximately 7500 L3 in a single event using a drenching syringe and blunt feeding needle. While grid counting of larvae is error-prone, volumetric aliquoting was used to ensure equitable distribution among each group of sheep. Severity of infection was assessed by fecal egg counts (McMasters), FAMACHA scoring via an investigator blinded to the treatment groups, and packed cell volume.
Animals were euthanized 50–60 days post-challenge using penetrating captive bolt in accordance with AVMA guidelines. Abomasum, spleen, lymph nodes, implants, and implant-associated lymphoid tissues were obtained for histopathology. The abomasal contents and mucosa were washed and the total number of adult parasites was enumerated. Total abomasal content was weighed and diluted and worms were removed and counted from three aliquots equivalent to 10% of total digesta. In addition, the mucosal surface was washed and all worms were counted. The total digesta count and the mucosa count were combined to achieve a total parasite count. When counting, immature adults or sex of worms was not differentiated.
H. contortus ELISA
Serum antibodies binding H. contortus antigens were measured by indirect ELISA. High-binding ELISA plates (Thermo Scientific) were coated overnight at 4 °C with vaccine antigen in PBS using 0.5 µg antigen/well, followed by washing in 5% non-fat dried milk blocking buffer. Duplicate dilutions of sheep serum were incubated for 1 h at room temperature and washed 3 times in PBS-T. Plates were then incubated with rabbit anti-sheep IgG-HRP (1:10,000) for 1 h in blocking buffer at room temperature. Plates were washed 3 times in PBS-T, developed with 1-Step™ Ultra TMB-ELISA Substrate Solution according to the manufacturer’s instructions, and stopped with 1 N hydrochloric acid. The absorbance of each well was read at 450 nm using a SpectraMax M2 microplate reader. Serum was diluted via 1:2 dilutions from 1:125 to 1:32,000 and the titer was determined as the last dilution with an absorbance greater than the same animal’s pre-bleed serum + 2 standard deviations at the same dilution. In addition, ELISA absorbance at a serum dilution of 1:500 was measured independently of titers.
Immunohistochemistry
Immunolocalization of antibody binding was determined on 5 μm thick paraffin sections of adult worms from the adjuvant group. Paraffin embedded slides were deparaffinized in xylene, and rehydrated with graded ethanol and distilled water. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide, and sections were then washed twice with PBS. Slides were boiled in 0.01 M citrate buffer (pH 9.0) for 2 min, placed in a steamer for 20 min and then kept at room temperature for 30 min. Non-specific binding sites were blocked with 5% non-fat dried milk for 30 min. Sections were incubated overnight with sheep serum pooled from all animals in each experimental group from the final blood sample, diluted 1:500 in blocking buffer, and washed 1× in PBS. Biotinylated rabbit anti-sheep antibody (Vectastain) was placed on the sections at a 1:500 dilution in blocking buffer for 30 min and washed with PBS. Sections were incubated with Vectastain® ABC Reagent diluted in 10 mL of PBS for 30 min and washed 3× with PBS. A compatible NovaRED® peroxidase substrate was added for 10 min and sections were washed in deionized-double distilled water. Slides were counterstained with hematoxylin, dehydrated through graded ethanol with a final wash in xylene, and mounted.
Statistical analysis
Statistical analysis was performed using Graphpad Prism or JMP. For each time point Dunnett’s multiple comparison was used to determine if the means of treatment groups were significantly different (P ≤ 0.05) from the values of the adjuvant-only group control (JMP Pro 14). Titer values were log2 transformed for the analysis.
