Three viruses were chosen for the production of antigens; two alphaviruses (CHIKV and MAYV) and one flavivirus (ZIKV). The VLPs were produced using the Invitrogen^TM Bac-to-Bac^® Baculovirus Expression System [1]. The baculovirus expression system is widely used in biotechnology for high yield recombinant protein expression by exploiting the life cycle of the virus in insect cells [2,3]. The advantage of using insect cells is the accurate post-translational modifications (phosphorylation, chaperone-assisted folding, glycosylation etc.) which cannot be achieved in E. coli.. To generate the CHIKV, ZIKV and MAYV VLPs, the genes encoding the viral structural proteins for the specific virus were placed downstream of the very strong polyhedrin promotor of the baculovirus, in this case Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus DNA is then used to transfect a Spodoptera frugiperda (Sf-21) cell line in order to produce recombinant baculoviruses expressing the VLPs.

Wageningen University & Research has constructs available for ZIKV and CHIKV [4], but not for MAYV. As the generation of such virus-like particles is similar, only the generation of MAYV VLPs will be described below.

Next to producing VLPs, strep-tagged surface glycoproteins of CHIKV were also produced. The benefit of these proteins is the ease of purification compared to VLPs, whilst offering comparable antigenic properties. The schematic structure of the surface glycoproteins is depicted in Figure 2 [5]. The native CHIKV polyprotein is cleaved into the individual E1 and E2 glcyoproteins, which are transported to the plasma membrane. There, they are taken up by budding nucleocapsids, resulting in mature, enveloped virions [6]. The introduction of a linker between E2 and E1 as well as the instruction of a strep-tag bypasses the need for membrane anchoring and yields secreted proteins, which are easily purified. The constructs for CHIKV secreted glycosylated trimeric E-protein were produced by a MSc student at the Wageningen University & Research. As with the production of the VLPs, the production of the Strep-tagged CHIKV-E uses the Bac-to-Bac^® system as well.

Figure 2: Schematic overview of CHIKV-E protein membrane organization of native and recombinant surface proteins, adapted from Voss et al. (2010).

Figure 3: Western blot of Sepharose-Strep-Tactin beads with CHIKV-E spikes. Fractions: pre, first flow through, last flow trough, last wash, elution 1-5.

As can be seen in Figure 3, the strep-tactin sepharose beads are capable of catching the strep-tagged CHIKV-E proteins. The experiment was repeated to cover a total of 6ml strep-tactin sepharose beads to be used in phage-panning later on.

The production of the secreted CHIKV-E construct is defined by Voss et al. and can be applied to Mayaro as well, as this virus is similar to chikungunya. For Zika, a different approach can be taken where the E protein present in the surface of the virion can be tagged [7]. Due to time constraints we chose not to pursue such methods and focus on the CHIKV-E proteins.