Over the last decades, several vaccine strategies enabling delivery of Ags for presentation by APC have been assayed with varying degrees of success [3
]. Since maturation of the DC is essential to trigger adaptive immune responses [29
], procedures that simultaneously target the antigen to DC and induce their maturation could lead to the development of a new generation of vaccines that might work in synergy with mild and safe adjuvants. We have previously reported that fusion of an antigen with EDA leads to antigen targeting to TLR4-expressing DC, enhancing cross-presentation and immunogenicity [4
]. Here, we describe a novel antigen delivery approach in which a biotinylated antigen is bound noncovalently to EDAvidin protein, a construct which retains the TLR4 targeting ability and inflammatory properties of EDA. This would allow the combination of the tetrameric EDAvidin with a broad range of commercially available antigens or adjuvants which can be easily biotinylated, facilitating the preparation of DC-targeted antigens to be used as immunogens for the induction of T cell responses.
By using different approaches, we have shown that EDAvidin forms tetrameric complexes and binds to biotinylated antigens with a very high affinity (Kd
~ 2.3 × 10−14
mol/L). Importantly, EDAvidin greatly increased biotinylated GFP uptake by DC and retained EDA proinflammatory capacity. It induced NF-κβ
activation, an important mediator for DC maturation [30
] and stimulated the production of TNF-α
by THP1 cells as well as the production of IL-12 by murine BMDC. The final aim when designing EDAvidin was to facilitate conjugation of EDA to biotinylated antigens to be used as vaccines. Thus, we compared the immunogenicity of EDAvidin plus NS3biot with that of a fusion protein between EDA and HCV NS3, an immunogen known to induce a specific T cell response when administered in the absence of additional adjuvants [19
]. We first found that EDAvidin interacted physically with NS3biot. But more importantly, we found that immunization with a mixture of EDAvidin and NS3biot induced a T cell immune response against NS3 similar to that obtained when using EDA-NS3 fusion protein. It is interesting to note that either when NS3 is not linked to the antigen delivery system (e.g., by using EDA instead of EDAvidin), or when tetrameric complexes do not retain the proinflammatory properties of EDA (e.g., when using streptavidin instead of EDAvidin), the immunogens show a much lower efficacy, demonstrating that both EDAvidin properties, antigen targeting, and DC activation are essential for efficient priming of T cells responses.
Multivalency of streptavidin and EDAvidin might be considered as an advantage for some applications. The tetrameric structure of EDAvidin might allow the combination of different biotinylated antigens or adjuvants within a single targeting vector. This would favour both sides required for antigen presentation, simultaneous targeting, and therefore induction of immune responses against several antigens, as well as the inclusion of new biotinylated adjuvants which would collaborate with EDA in APC activation [17
], a situation that has been demonstrated to improve phagosome maturation and antigen presentation by APC [32
Proteins can be biotinylated chemically or enzymatically using already established protocols. Using our strategy, we could also consider the possibility to biotinylate more complex antigenic structures such as whole cells for their engineering to codisplay immunomodulatory molecules, as it has been described previously with a different approach [33
]. EDAvidin could also be used to decorate a biotinylated tumor cell to render it more immunogenic allowing its capture by TLR4 expressing DC. Future experiments need to be conducted to explore this possibility.
In summary, we have found that a chimeric protein containing EDA fused to the N terminus of streptavidin retains functional properties of EDA and facilitates its conjugation to any antigen of choice and results in a new tool which opens a new way to use this antigen delivery system in vaccination against infectious diseases and cancer.