Oral Immunogenicity in Mice and Sows of Enterotoxigenic Escherichia Coli Outer-Membrane Vesicles Incorporated into Zein-Based Nanoparticles

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of illness and death in neonatal and recently weaned pigs. The immune protection of the piglets derives from maternal colostrum, since this species does not receive maternal antibodies through the placenta. In the present study, outer membrane vesicles (OMVs) obtained from main ETEC strains involved in piglet infection (F4 and F18 serotypes), encapsulated into zein nanoparticles coated with Gantrez®® AN-mannosamine conjugate, were used to orally immunize mice and pregnant sows. Loaded nanoparticles were homogeneous and spherical in a shape, with a size of 220–280 nm. The diffusion of nanoparticles through porcine intestinal mucus barrier was assessed by a Multiple Particle Tracking technique, showing that these particles were able to diffuse efficiently (1.3% diffusion coefficient), validating their oral use. BALB/c mice were either orally immunized with free OMVs or encapsulated into nanoparticles (100 µg OMVs/mouse). Results indicated that a single dose of loaded nanoparticles was able to elicit higher levels of serum specific IgG1, IgG2a and IgA, as well as intestinal IgA, with respect to the free antigens. In addition, nanoparticles induced an increase in levels of IL-2, IL-4 and IFN-γ with respect to the administration of free OMVs. Orally immunized pregnant sows with the same formulation elicited colostrum-, serum- (IgG, IgA or IgM) and fecal- (IgA) specific antibodies and, what is most relevant, offspring suckling piglets presented specific IgG in serum. Further studies are needed to determine the infection protective capacity of this new oral subunit vaccine

Protective Passive Immunity in Escherichia coli ETEC-Challenged Neonatal Mice Conferred by Orally Immunized Dams with Nanoparticles Containing Homologous Outer Membrane Vesicles

“…Vaccination of pregnant mothers has demonstrated to be a good approach to control endemic diseases [23]. The results on protection presented here together with those reported previously on immunogenicity in sows [13] support further studies of immunization with OMV into nanoparticles during pregnancy [34,35].…”

“…Isolated OMV obtained from ETEC strain in terms of morphology such as the range size were confirmed with previous reports [13] showing to be spherical (15-200 nm) without cell debris. The yield obtained in the process of isolating OMV was 23.0 ± 0.1 µg/mg, referred to the original cell culture wet weight.…”

“…The characterization studies of the OMV used in this work revealed the presence of relevant immunogenic components such as flagellin, OmpC/OmpA, lipoproteins and LPS, among others [15][16][17]. The existence of OMV on the surface of nanoparticles and the presence of YghJ, which degrades the major mucins MUC2 and MUC3 [18], could explain the capability of nanoparticles containing OMV to diffuse in the intestinal mucus as described previously [13].…”

“…In addition, the use of Gantrez ® AN as the material to produce nanoparticles and/or polymer conjugates may be of interest to promote strong and balanced immune responses due to its capability as agonist for TLR2 and TLR4 [10]. The immunostimulant effect of Gantrez ® AN formulations was reported in vaccination studies against Salmonella enterica [30] or Shigella flexneri in mice [31], and vaccinated sows with this particular OMV-GM-Zein nanoparticle vaccine formulation showed specific IgG, IgA and IgM in serum [13]. In view of these results, we finally evaluated the capacity of this new vaccine to confer protection to offspring from vaccinated mothers.…”

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“…IL-4 promotes IgG1 conversion, expanding the proportion of B cells producing IgG1, and IFN-γ inhibits IgG1 but stimulates IgG2a transformation. 39 In this work, to ensure that the quantities of drugs in the tumor were equal between groups and to achieve sufficient activity with FUS, we injected equal quantities of the designed drugs locally into the tumor, followed by FUS exposure the next day. Thus, the concentration of the drugs was limited in in vivo circulation, which may make a difference in the immune response from administration by caudal injection.…”