Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.
Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C). Expression levels and assembly into empty capsids were analyzed in insect cells and larvae. We observed that the use of the single-promoter vector allowed higher levels of expression both in insect cells and larvae. Recombinant capsid proteins produced by both vectors were recognized by monoclonal antibodies (mAbs) directed against conformational epitopes of FMDV A/Arg/01 and proved to self-assemble into empty capsids (75S) and pentamers (12S) when analyzed by sucrose gradient centrifugation.
The presence and molecular characterization of Clostridium perfringens in healthy Merino lambs over a six-month period was investigated in this study. Overall, a high prevalence of C. perfringens was detected, even in day-old lambs. Even though the majority of the isolates were characterized as being of type A, types C and D were also isolated. Furthermore, a high genetic diversity was observed by PFGE among the type A isolates.
Inactivated Foot-and-Mouth Disease (FMD) vaccine has proven to be effective in the control of the disease. However, its production has some disadvantages, including the costly biosafety facilities required for the production of huge amounts of growing live virus, the need of an exhaustive purification process to eliminate non-structural proteins of the virus in the final formulations in order to differentiate infected from vaccinated animals and variable local regulatory restrictions to produce and commercialize the vaccine. Thus, a novel vaccine against FMD that overcome these restrictions is desirable. Although many developments have been made in this regard, most of them failed in terms of efficacy or when considering their transferability to the industry. We have previously reported the use of transient gene expression in mammalian cells to produce FMD virus-like particles (VLPs) as a novel vaccine for FMD and demonstrated the immunogenicity of the recombinant structures in animal models. Here, we report the optimization of the production system by assaying different DNA:polyethylenimine concentrations, cell densities, and direct and indirect protocols of transfection. Also, we evaluated the reproducibility and scalability of the technology to produce high yields of recombinant VLPs in a cost-effective and scalable system compatible with industrial tech-transfer of an effective and safe vaccine.
Mucosal epithelia are primary sites for antigen entry. The microenvironment in these mucosal barriers has a marked influence on the immune response that ultimately ensues (1) . The bioactive compounds present in foods would have an impact on the immunocompetence, especially in the intestinal villi (IV). In this way, the attention is focused on the possibility of modulating the immune response for enhancing health and quality life.Allergy problems due to dietary factors were increased during the last few years almost at epidemic levels (2) . It is known that polyphenols have some effects related to immune response but results are partial and controversial. Immunoglobulin E (IgE) has a central role in allergic responses and secretory immunoglobulin A (SIgA) is important to generate immune protection without inflammation.Onion is a food that presents a remarkable combination of bioactive compounds with high content of Quercetin (polyphenol). The aim of this study was to evaluate ex vivo the effect of consumption of onion or pure Quercetin on cellular immune responses in IV. An experimental model was used to evaluate the effect of protein malnutrition and allergy response. Weanling rats of wistar strain were fed a protein-free diet until they lost 25 % of their initial body weight. Re-feeding was performed by the administration of an experimental diet containing 20 % casein as the only source of protein (re-nourished group = R). Other experimental groups received this experimental diet plus onion juice (R + O) in quantity equivalent to normal consumption of an adult man or Quercetin (R + Q) in quantity equivalent to that containing in R + O, both added to drinking water during 40 days. Three well-nourished groups were used as normal controls (C) which were fed with standard commercial diet or the same diet plus onion juice (C + O) or plus Quercetin (C + Q). The small Intestine was removed and processed by Saint-Marie's technique. IgA + and IgE + B cells, CD5 + T-cells and CD4 + T sub-population in IV were assessed by indirect immunofluorescence technique. The animal protocol was approved by the ethical committee of the University of Buenos Aires and all procedures were in accordance with the department's guide for the care and use of laboratory animals. Results showed: (1) T lymphocytes, CD4 helper sub-population on intestinal Lamina Propria showed no significant difference. Instead, CD5 population showed significant difference (P = 0.047) for R: (mean; SE) 193; 11 compared to C: 227; 12 and within the R groups for R + Q: 153; 19 compared to R: 200; 19 (P = 0.018). This reveals that Quercetin consumption reduced CD5 T-cells. (2) IgA + B lymphocytes showed no significant difference in R related to onion or Quercetin consumption (R + O, R + Q) but they presented significantly lower levels in R: 188; 12 compared to C: 254; 13 (P = 0.001). (3) IgE + B cells were increased in R: 165; 7 as compared to C: 62; 7 (P > 0.0001) probably due to the administration of the experimental diet in this model of malnutrition. The IgE +...
Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.
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