Kinetics of foot-and-mouth disease vaccine-induced antibody responses in buffaloes (Bubalus bubalis): avidity ELISA as an alternative to the virus neutralization test.

The role of water buffaloes in foot-and-mouth disease (FMD) epidemiology as one of the major hosts of the virus that can develop persistent asymptomatic infection highlights the importance of sustaining surveillance on the antibody response elicited by vaccination in these animals. There is gap in the knowledge on how serological assays that measure antibodies against capsid proteins perform with buffalo samples and which would be the most reliable test to substitute the virus neutralization test (VNT) a cumbersome and low-throughput tool for field surveillance. Alternatively, the liquid-phase blocking sandwich ELISA (LPBE) is commonly used. Previous data from our laboratory demonstrated that the vaccine-induced antibodies assessed by the LPBE yielded low specificity with buffaloes' samples. In contrast, a single-dilution avidity ELISA (AE) aimed to detect high-avidity antibodies against exposed epitopes, combined with an indirect ELISA (IE) to assess IgG levels, produced more reliable results. Here we analyzed for the first time the kinetics of the antibodies induced by vaccination in two different buffalo herds (n = 91) over 120 days using AE, IE, LPBE, and the VNT. Kinetics were similar in the different assays, with an increase of antibodies between 0- and 14-days post-vaccination (dpv) which were maintained thereafter. VNT and AE results were concordant (Kappa value = 0.76), and both assays revealed a decay in the antibody response in calves with maternal antibodies at 90 and 120 dpv, which was not evidenced by the LPBE. These results show that kinetics of antibody responses to FMD vaccination are similar in buffalo and cattle, and support the use of indirect ELISA assays, in particular Avidity ELISA, as alternatives to the VNT for vaccine-immunity monitoring irrespectively of the animal's passive or active immune status.

Assessment on Different Vaccine Formulation Parameters in the Protection against Heterologous Challenge with FMDV in Cattle.

Foot-and-mouth disease (FMD) remains one of the major threats to animal health worldwide. Its causative agent, the FMD virus (FMDV), affects cloven-hoofed animals, including farm animals and wildlife species, inflicting severe damage to the international trade and livestock industry. FMDV antigenic variability remains one of the biggest challenges for vaccine-based control strategies. The current study analyzed the host's adaptive immune responses in cattle immunized with different vaccine protocols and investigated its associations with the clinical outcome after infection with a heterologous strain of FMDV. The results showed that antigenic payload, multivalency, and revaccination may impact on the clinical outcome after heterologous challenge with FMDV. Protection from the experimental infection was related to qualitative traits of the elicited antibodies, such as avidity, IgG isotype composition, and specificity diversity, modulating and reflecting the vaccine-induced maturation of the humoral response. The correlation analyses of the serum avidity obtained per vaccinated individual might suggest that conventional vaccination can induce high-affinity immunoglobulins against conserved epitopes even within different FMDV serotypes. Cross-reaction among strains by these high-affinity antibodies may support further protection against a heterologous infection with FMDV.

Enzymatic Functionalization of Wood as an Antifouling Strategy against the Marine Bacterium Cobetia marina.

The protection of wood in marine environments is a major challenge due to the high sensitivity of wood to both water and marine microorganisms. Besides, the environmental regulations are pushing the industry to develop novel effective and environmentally friendly treatments to protect wood in marine environments. The present study focused on the development of a new green methodology based on the laccase-assisted grafting of lauryl gallate (LG) onto wood to improve its marine antifouling properties. Initially, the enzymatic treatment conditions (laccase dose, time of reaction, LG concentration) and the effect of the wood specie (beech, pine, and eucalyptus) were assessed by water contact angle (WCA) measurements. The surface properties of the enzymatically modified wood veneers were assessed by X-ray photoelectron spectroscopy (XPS), Fourier transform-infrared spectroscopy (FTIR). Antifouling properties of the functionalized wood veneers against marine bacterium Cobetia marina were studied by scanning electron microscopy (SEM) and protein measurements. XPS and FTIR analysis suggested the stable grafting of LG onto the surface of wood veneers after laccase-assisted treatment. WCA measurements showed that the hydrophobicity of the wood veneers significantly increased after the enzymatic treatment. Protein measurements and SEM pictures showed that enzymatically-hydrophobized wood veneers modified the pattern of bacterial attachment and remarkably reduced the bacterium colonization. Thus, the results observed in the present study confirmed the potential efficiency of laccase-assisted treatments to improve the marine antifouling properties of wood.

Nanocellulose-Based Inks-Effect of Alginate Content on the Water Absorption of 3D Printed Constructs.

2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) oxidized cellulose nanofibrils (CNF) were used as ink for three-dimensional (3D) printing of porous structures with potential as wound dressings. Alginate (10, 20, 30 and 40 wt%) was incorporated into the formulation to facilitate the ionic cross-linking with calcium chloride (CaCl2). The effect of two different concentrations of CaCl2 (50 and 100 mM) was studied. The 3D printed hydrogels were freeze-dried to produce aerogels which were tested for water absorption. Scanning Electronic Microscopy (SEM) pictures demonstrated that the higher the concentration of the cross-linker the higher the definition of the printed tracks. CNF-based aerogels showed a remarkable water absorption capability. Although the incorporation of alginate and the cross-linking with CaCl2 led to shrinkage of the 3D printed constructs, the approach yielded suitable porous structures for water and moisture absorption. It is concluded that the 3D printed biocomposite structures developed in this study have characteristics that are promising for wound dressings devices.

3D Printing High-Consistency Enzymatic Nanocellulose Obtained from a Soda-Ethanol-O2 Pine Sawdust Pulp.

Soda-ethanol pulps, prepared from a forestry residue pine sawdust, were treated according to high-consistency enzymatic fibrillation technology to manufacture nanocellulose. The obtained nanocellulose was characterized and used as ink for three-dimensional (3D) printing of various structures. It was also tested for its moisture sorption capacity and cytotoxicity, as preliminary tests for evaluating its suitability for wound dressing and similar applications. During the high-consistency enzymatic treatment it was found that only the treatment of the O2-delignified pine pulp resulted in fibrillation into nano-scale. For 3D printing trials, the material needed to be fluidized further. By 3D printing, it was possible to fabricate various structures from the high-consistency enzymatic nanocellulose. However, the water sorption capacity of the structures was lower than previously seen with porous nanocellulose structures, indicating that further optimization of the material is needed. The material was found not to be cytotoxic, thus showing potential as material, e.g., for wound dressings and for printing tissue models.

3D Printable Filaments Made of Biobased Polyethylene Biocomposites.

Two different series of biobased polyethylene (BioPE) were used for the manufacturing of biocomposites, complemented with thermomechanical pulp (TMP) fibers. The intrinsic hydrophilic character of the TMP fibers was previously modified by grafting hydrophobic compounds (octyl gallate and lauryl gallate) by means of an enzymatic-assisted treatment. BioPE with low melt flow index (MFI) yielded filaments with low void fraction and relatively low thickness variation. The water absorption of the biocomposites was remarkably improved when the enzymatically-hydrophobized TMP fibers were used. Importantly, the 3D printing of BioPE was improved by adding 10% and 20% TMP fibers to the composition. Thus, 3D printable biocomposites with low water uptake can be manufactured by using fully biobased materials and environmentally-friendly processes.

Green Binder Based on Enzymatically Polymerized Eucalypt Kraft Lignin for Fiberboard Manufacturing: A Preliminary Study.

The capability of laccase from Myceliophthora thermophila to drive oxidative polymerization of Eucalyptus globulus Kraft lignin (KL) was studied as a previous step before applying this biotechnological approach for the manufacturing of medium-density fiberboards (MDF) at a pilot scale. This method, which improves the self-bonding capacity of wood fibers by lignin enzymatic cross-linking, mimics the natural process of lignification in living plants and trees. An interesting pathway to promote these interactions could be the addition of lignin to the system. The characterization of E. globulus KL after enzymatic treatment showed a decrease of phenolic groups as well as the aromatic protons without loss of aromaticity. There was also an extensive oxidative polymerization of the biomolecule. In the manufacture of self-bonded MDF, the synergy generated by the added lignin and laccase provided promising results. Thus, whenever laccase was present in the treatment, MDF showed an increase in mechanical and dimensional stability for increasing amounts of lignin. In a pilot scale, this method produced MDF that meets the requirements of the European standards for both thickness swell (TS) and internal bonding (IB) for indoor applications.

Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle.

Serological assessment of the heterologous response among Foot-and-Mouth Disease Virus (FMDV) strains is mainly performed by virus neutralization test (VNT), liquid phase blocking ELISA and complement fixation assay. In this study two high-throughput ELISA techniques, avidity and IgG subtype ELISA, were developed and used to further characterize heterologous antibody responses in cattle during vaccination and challenge. Both assays were applied to a set of previously characterized sera from animals immunized with an inactivated A24 Cruzeiro/Brazil/55 (A24 Cruzeiro) strain monovalent FMDV vaccine and challenged with the heterologous A/Argentina/2001 (A/Arg/01) strain. Single dilution avidity ELISA assessment showed that animals that were protected against A/Arg/01 challenge had higher avidity antibodies to this heterologous strain than non-protected cattle. Animals with low or even undetectable anti-A/Arg/01 serum-neutralizing titers that passed the heterologous challenge presented higher IgG1/IgG2 ratio than non-protected animals. In this study, the three assessments (VNT and both ELISAs) discriminated between protected and not protected animals against a heterologous challenge. The combination of these techniques may be applied to complement current indirect serological vaccine-matching assessments. The measurement of these qualitative parameters may provide additional information to understand the mechanisms underlying FMD heterologous responses and the induction of cross-protection in cattle.

Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle.

Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.

Strategies for improving vaccine antigens expression in transgenic plants: fusion to carrier sequences.

Transgenic plants are gaining increasing attention from the industry as a natural bioreactor for the production of industrial and chemical products. Optimization of transgene expression in plant cells holds the key to maximizing the potential of plants for producing proteins of commercial interest. This chapter is devoted to the description of the methods utilized for the generation of transgenic plants expressing a canine parvovirus vaccine peptide or virus-like particles from a rabbit calicivirus.

Multimerization of peptide antigens for production of stable immunogens in transgenic plants.

Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12mug/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the beta-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity.

Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants.

Vaccines produced in transgenic plants constitute a promising alternative to conventional immunogens, presenting the possibility of stimulating secretory and systemic immunity against enteric pathogens when administered orally. Protection against enteric pathogens affecting newborn animals requires, in most cases, the stimulation of lactogenic immunity. Here, the group presents the development of an experimental immunogen based on expression of an immunorelevant peptide, eBRV4, of the VP4 protein of bovine rotavirus (BRV), which has been described as harbouring at least one neutralizing epitope as well as being responsible for the adsorption of the virus to epithelial cells. The eBRV4 epitope was efficiently expressed in transgenic alfalfa as a translational fusion protein with the highly stable reporter enzyme beta-glucuronidase (betaGUS), which served as a carrier, stabilized the synthesized peptide and facilitated screening for the higher expression levels in plants. Correlation of expression of the eBRV4 epitope in plants with those presenting the highest betaGUS activities was confirmed by a Western blot assay specific for the BRV peptide. The eBRV4 epitope expressed in plants was effective in inducing an anti-rotavirus antibody response in adult female mice when administered either intraperitoneally or orally and, more importantly, suckling mice born from immunized female mice were protected against oral challenge with virulent rotavirus. These results demonstrate the feasibility of inducing lactogenic immunity against an enteric pathogen using an edible vaccine produced in transgenic plants.