A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1.

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Bovine Dendritic Cell Activation, T Cell Proliferation and Antibody Responses to Foot-And-Mouth Disease, Is Similar With Inactivated Virus and Virus Like Particles.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses in the livestock industry. Currently available vaccines are based on the inactivated FMD virus (FMDV). Although inactivated vaccines have been effective in controlling the disease, they have some disadvantages. Because of these disadvantages, investigations are being made to produce vaccines in low containment facilities. The use of recombinant empty capsids (also referred as Virus Like Particles, VLPs) has been reported to be a promising candidate as a subunit vaccine because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. Mignaqui and collaborators have produced recombinant FMDV empty capsids from serotype A/ARG/2001 using a scalable technology in mammalian cells that elicited a protective immunity against viral challenge in a mouse model. However, further evaluation of the immune response elicited by these VLPs in cattle is required. In the present work we compare the effect that VLPs or inactivated FMDV has on bovine dendritic cells and the humoral response elicited in cattle after a single vaccination.

Foot-and-Mouth Disease: Optimization, Reproducibility, and Scalability of High-Yield Production of Virus-Like Particles for a Next-Generation Vaccine.

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.

Advances in novel vaccines for foot and mouth disease: focus on recombinant empty capsids.

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.

Transient gene expression in serum-free suspension-growing mammalian cells for the production of foot-and-mouth disease virus empty capsids.

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.