Layered Double Hydroxides (LDH) as Delivery Vehicles of a Chimeric Protein Carrying Epitopes from the Porcine Reproductive and Respiratory Syndrome Virus

Abstract

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) causes reproductive failure and respiratory symptoms, leading to huge economic losses for the pig farming industry. Although several vaccines against PRRSV are available in the market; they show an overall low efficacy, and several countries have the need for vaccines covering the local, circulating variants. This project aims at developing a new chimeric antigen targeting specific epitopes from PRRSV and evaluating two test adjuvants to formulate a vaccine candidate. The test antigen was called LTB-PRRSV, which was produced recombinantly in Escherichia coli and consisted of the heat labile enterotoxin B subunit from E. coli (LTB) and four epitopes from PRRSV. LTB-PRRSV was rescued as inclusion bodies and methods for its solubilization, IMAC-based purification, and refolding were standardized, leading to mean yields of 18 mg of pure protein per liter culture. Layered double hydroxides (LDH) have been used as vaccine adjuvants given their biocompatibility, low cost, and positive surface charge that allows an efficient adsorption of negatively charged biomolecules. Therefore, LDH were selected as delivery vehicles of LTB-PRRSV. Pure LTB-PRRSV was adsorbed onto LDH by incubation at different LDH:LTB-PRRSV mass ratios (1:0.25, 1:0.5, 1:1, and 1:2) and at pH 9.5. The best adsorption occurred with a 1:2 mass ratio, and in a sucrose-tween solution. The conjugates obtained had a polydispersity index of 0.26, a hydrodynamic diameter of 192 nm, and a final antigen concentration of 64.2 μg/mL. An immunogenicity assessment was performed by injecting mice with LDH:LTB-PRRSV, Alum/LTB-PRRSV, or LTB-PRRSV in a scheme comprising three immunizations at two-week intervals and two dose levels (1 and 5 μg). LTB-PRRSV was capable of inducing strong humoral responses, which lasted for a longer period when LDH was used as the delivery vehicle/adjuvant. The potential of LDH to serve as an attractive carrier for veterinary vaccines is discussed.

Keywords: adjuvant; humoral response; multi-epitopic vaccine; nano-vaccine; nanoparticle.

Figure 1

SDS–PAGE analyses of soluble and insoluble protein fractions during the production of LTB–PRRSV (16 kDa). (A) Soluble fractions. MWM, molecular weight marker; 1, soluble fraction from colony 1; 2, soluble fraction from colony 2; 3, soluble fraction from colony 3; 4, 5, and 6, duplicates of soluble fractions from colonies 1, 2, and 3, respectively. (B) Insoluble fractions. MWM, molecular weight marker; 1 insoluble fraction from colony 1; 2, insoluble fraction from colony 2; 3, insoluble fraction from colony 3; 4, 5, and 6, duplicates of insoluble fractions from colonies 1, 2, and 3, respectively.

Figure 2

Extracts of LTB–PRRSV solubilized with urea solutions at different concentrations. Lane 1, LTB–PRRSV solubilized during 30 min with a 4 M urea solution; Lane 2, LTB–PRRSV solubilized during 30 min with an 8 M urea solution; Lane 3, LTB–PRRSV solubilized overnight with an 8 M urea solution.

Figure 3

Fractions from the LTB–PRRSV IMAC-based purification. Lane MWM, molecular weight marker; Lanes 1–8, unbound fractions collected upon column washing. Fraction 8 was used for the refolding step, having a 92% purity.

Figure 4

Layered double hydroxide (LDH) NP synthesized and then conjugated with recombinant LTB–PRRSV. (A) Size distributions during consecutive stages of refinement after their synthesis by coprecipitating a 3:1 molar ratio of Mg²⁺:Al³⁺. Washing twice prior (pre-HT) their hydrothermal treatment (HT) and washing once after this (post-HT) results in monodispersed LDH NP. Insert: TEM image of a typical final suspension. (B) Size evolution of LTB–PRRSV/LDH after the physisorption of increasing amounts of antigen protein with mass ratios from 1:0.5 to 1:2 during 1 h at room temperature. Insert: PAGE image of antigen adsorbed on LDH. Hydrodynamic diameter (d_Z), polydispersity index (PdI), and ζ potential of LDH synthesized and suspended in water, LDH suspended in 20 mM Tris (pH 9.5), and of LDH conjugated with antigen (LDH/ag mass ratios of 1:0.5, 1:1, 1:2) and suspended in 10% sucrose containing 0.01% Tween 20.

Figure 5

Humoral response induced by the LTB–PRRSV antigen using different formulations. Mice groups (n = 4) were s.c. injected on days 0, 14, and 28 with 5 µg of LTB–PRRSV alone (LTB–PRRSV), 5 µg of LTB–PRRSV/LDH conjugate (LTB–PRRSV:LDH), 5 µg of LTB–PRRSV + Al(OH)₃ (LTB–PRRSV:Alum), or the vehicle alone (Vehicle). Blood samples were withdrawn on days 42 (A) and 86 (B) to measure seric anti-LTB–PRRSV IgG levels by ELISA. The asterisks denote statistical differences (p < 0.05) versus the LTB–PRRSV/Alum group as control.