Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Production of functional active human growth factors in insects used as living biofactories.

Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications.

Antibodies against Marinobacter algicola and Salmonella typhimurium flagellins do not cross-neutralize TLR5 activation.

Flagellins evoke strong innate and adaptive immune responses. These proteins may play a key role as radioprotectors, exert antitumoral activity in certain types of tumor and reduce graft-versus-host disease in allogeneic hematopoietic stem cell transplant recipients. Notwithstanding, flagellins are highly immunogenic, and repeated use leads to their neutralization by systemic antibodies. This neutralization is not prevented by using functional deleted flagellins. These observations led us to explore the possibility of preventing initial neutralization by means of another functional flagellin that does not belong to common pathogenic bacteria but that has the capacity to activate TLR5. Here we characterized the functional capacity of the two-phase Marinobacter algicola (MA)-derived flagellins (F and FR) as systemic and mucosal adjuvants and compared their performance with that of Salmonella typhimurium (STF) flagellins (FljB and FliC). We also report for the first time on the in vitro and in vivo capacity of various flagellins to trigger TLR5 activation in the presence of species-specific anti-flagellin antibodies, the cross-neutralization mediated by these antibodies, and the sequential use of these flagellins for TLR5 activation. Our results showed that MA flagellins behave in a similar way to STF ones, inducing pro-inflammatory cytokines (IL8, CCL20, CCL2) and evoking a strong in vivo antibody response against a model epitope. More importantly, MA flagellins were fully functional, in vitro or in vivo, in the presence of a high concentration of neutralizing anti-flagellin STF antibodies, and STF flagellin was not inhibited by neutralizing anti-flagellin MA antibodies. The use of active flagellins from distinct bacteria could be a useful approach to prevent systemic neutralization of this group of adjuvants and to facilitate the rational design of flagellin-based vaccines and/or other therapeutic treatments (against ischemia, acute renal failure, tumors, ionizing radiations and also to improve the outcome of bone marrow transplants).