Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

Abstract

BackgroundChikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively.ResultsExpression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits.ConclusionsChikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

Prime-Boost Immunization Strategies against Chikungunya Virus

“…Several CHIKV vaccine candidates are under development (9), including attenuated (10)(11)(12)(13)(14)(15)(16) or inactivated (17)(18)(19) CHIKV, alphavirus chimeras (20)(21)(22), and subunit (23)(24)(25)(26)(27) and genetic (21,(28)(29)(30)(31) vaccines. Moreover, we have reported previously on the construction and preclinical evaluation of novel CHIKV vaccine candidates, based on attenuated CHIKV (12) or recombinant modified vaccinia virus Ankara (MVA) expressing CHIKV antigens (MVA-CHIKV) (29), that were able to induce strong immunogenicity and efficacy in a mouse model.…”

Novel Attenuated Chikungunya Vaccine Candidates Elicit Protective Immunity in C57BL/6 mice

“…A number of CHIKV vaccine candidates have been described, including attenuated (11)(12)(13) or inactivated (14,15) CHIKV, alphavirus chimeras (16), and subunit (17)(18)(19)(20) and genetic (21)(22)(23)(24) vaccines. Since inactivated, subunit, and genetic vaccines typically require several immunizations to confer immunity and are expensive to produce, live-attenuated viruses are usually the most potent vaccines because of their ability to infect cells and stimulate both innate and adaptive immune responses, usually without the need for a booster immunization (25).…”

Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response

“…After autocatalytic cleavage of the capsid protein, the remaining polyprotein translocates to the ER. In the ER, the polyprotein is cleaved by host signalases into precursors E2 and E1, which are N-linked glycosylated before being transported to the Golgi network (Jose et al, 2009;Metz et al, 2011). We investigated whether CHIKV envelope glycoproteins induced the UPR and whether CHIKV replication affected UPR target gene expression and the expression of ATF4.…”

“…Membranes were blocked in 3 % skimmed milk in PBS with 0.05 % Tween 60 (PBST) for 1 h at room temperature. Membranes were washed three times for 5 min each with PBST and subsequently incubated for 1 h at room temperature with rabbit polyclonal anti-E2 (diluted 1 : 20 000; Metz et al, 2011), anti-P-eIF2a (diluted 1 : 500; Abcam) and anti-b-tubulin (diluted 1 : 4000; Abcam) in PBST, respectively. Membranes were washed and treated with alkaline phosphatase-conjugated goat anti-rabbit IgG mAb (Sigma), diluted 1 : 3000 in PBST, for 45 min at room temperature.…”