Immunogenic recombinant Mayaro virus-like particles present natively assembled glycoprotein

Abstract

Virus-like particles (VLPs) are an established vaccine platform and can be strong immunogens capable of eliciting both humoral and cellular immune responses against a range of pathogens. Here, we show by cryo-electron microscopy that VLPs of Mayaro virus, which contain envelope glycoproteins E1-E2 and capsid, exhibit an architecture that closely resembles native virus. In contrast to monomeric and soluble envelope 2 (E2) glycoprotein, both VLPs as well as the adenovirus and modified vaccinia virus Ankara (MVA) vaccine platforms expressing the equivalent envelope glycoproteins E1-E2, and capsid induced highly neutralising antibodies after immunisation. The levels of neutralising antibodies elicited by the viral-vectored vaccines of structural proteins and VLPs increased significantly upon boosting. Immunisation of Mayaro virus VLPs in mice with or without an adjuvant (poly:IC) yielded similar levels of neutralising antibodies suggesting that the VLPs may be used for immunisation without the need for an adjuvant. A single or two doses of non-adjuvanted 5 µg of MAYV VLP vaccination provided significant protection against viremia and MAYV-induced foot swelling in the C57BL/6 mouse challenge model. MAYV VLPs represent a non-infectious vaccine candidate, which may constitute a complementary option for future immunisation strategies against this important emerging alphavirus.

Fig. 1. Production, purification and characterisation of MAYV VLP.

a A schematic representation of the MAYV genome and the structural polyprotein (C, E3, E2, 6K, and E1). b Western blot analysis of MAYV VLP production in mammalian cells. The supernatant from HEK293T cells containing VLP was concentrated using 20% sucrose cushion centrifugation prior to WB analysis. WB using mice sera vaccinated with ChAdOx1 May (sMAYV) or Chik (sCHIKV) in reduced (R) and non-reduced (NR) conditions. c WB analysis of MAYV VLP following subsequent sucrose gradient purifications compared to recombinant MAYV E2 antigen (without the TM domain) using ChAdOx1 sMAYV. Wells 1 = MAYV VLP after sucrose cushion, 2 = non-infected (mock), 3/4 = MAYV VLP after sucrose gradient top band (3) and bottom band (4), rE2 = recombinant MAYV E2. d SDS-PAGE gel (Coomassie staining) and WB of purified MAYV VLP using ChAdOx1 sMAYV vaccinated mice sera under reducing conditions are shown. e Transmission electron microscopy (negative stain) images of purified MAYV VLPs are shown.

Fig. 2. Cryo-EM structure of MAYV VLP to 4.5 Å resolution and compositional analysis of N-linked glycosylation presented by MAYV VLP glycoproteins.

a Structure of MAYV-VLP seen along the two-fold axis of icosahedral symmetry. b The same view as in (a), with frontmost E1/E2 glycoprotein spikes removed to reveal the capsid. c A central slab of density is shown. d One asymmetric unit is shown orthogonally. The symmetric unit is shown rotated 90 degrees around the axis indicated. e The structural comparison of our MAYV VLP coloured in lime to mature live MAYV virion in orange (EMDB-22961) and its cross-section display (f). (g) N-linked glycosylation of purified MAYV VLP produced in HEK293T cells was assessed by WB under reducing condition using mice sera vaccinated with ChAdOx1 sMAYV, VLP, E1 and E2 following digestion with PNGase F or Endo-Hf. h N-glycan analysis by in-line liquid chromatography-mass spectrometry of glycopeptides. Green—oligomannose-type glycan, pink—complex-type glycan, pink = hybrid type glycans. Complex glycans are grouped according to the number of antennae and presence of core fucosylation (A1 to FA4).

Fig. 3. Comparative assessment of MAYV vaccine platforms in BABL/c strain of mice.

a BALB/c mice (n = 6) were immunised intramuscularly (IM) at the intervals as outlined above and the timepoints of blood serum sampling for ELISA/ELISpot are shown. The name of the vaccination group, priming and boosting agents are shown. b–e Humoral immune responses elicited by MAYV VLP, viral-vectored vaccines and E1/E2 sub-unit vaccine. MAYV E1 or E2 ELISA was performed with sera obtained from vaccinated mice at week 6 after a single vaccination, 2 weeks and 8 weeks after P-B vaccination and 4 weeks and 22 weeks after P-B-B vaccination (b, d). Anti-MAYV E1 or E2 ELISA titres were calculated for all groups as shown in (b–e). The kinetics of anti-MAYV E1 or E2 titres over time are shown in (c, e). Turkey’s multiple comparisons test was used to analyse the groups (b, d). (f, g) Cellular immune responses elicited by MAYV VLP, viral-vectored vaccines and E1/E2 sub-unit vaccines. Peripheral blood mononuclear cells (PBMCs) were cultured with a peptide pool containing the MAYV structural antigens by ELISpot for IFNγ producing cells using mice serum; (f) 3 weeks post-prime (g) 4 weeks post-prime-boost-boost (PBB) vaccinations. Values represent the spot-forming cells (SFC) per million PBMCs. 20-mer peptides spanning the MAYV structural polyprotein (10 µg/mL) were used for stimulation. P values in were determined by one-way ANOVA and Tukey’s multiple comparisons test. Line colours represent mice vaccinated with each vaccine. The mean antibody titres or SFCs and error bars are shown by solid colour lines.

Fig. 4. Neutralisation potency of MAYV VLP vaccinated mice sera.

a–c MAYV and CHIKV VRP neutralisation assays. MAYV NT₅₀ titres for BALB/c mice serum samples post immunisations with MAYV VLP, viral-vectored sMAYV or MAYV E2 are shown. d PRNT assay showing percentage neutralisation of mice serum vaccinated with MAYV VLP, MAYV E2 or control (ZIKV Envelope) 4 weeks post PBB. PRNT50, 60, 80 and 90 titres for each group are shown in the table. Coloured lines represent the mean with SD and error bars. P values in (b, c) were determined by one-way ANOVA and Tukey’s multiple comparisons test. p > 0.05 (ns), p < 0.01 (**), p < 0.0001 (****). P value in (c) was determined by the Mann-Whitney U test. p > 0.05 (ns).

Fig. 5. Assessment of protective efficacy induced by MAYV VLP vaccines.

a MAYV VLP (either prime only or prime boost) vaccinated C57BL/6 mice were inoculated subcutaneously in the right hind footpad with 5 × 10⁶ PFU MAYV at 4 weeks post-immunisation. After MAYV challenge, viraemia was monitored daily from 3-day post-infection (dpi) until 10 dpi. Joint swelling of the footpad was scored starting at day 0 and after from 3 to 10 dpi. b. viral load kinetics of MAYV challenge groups were monitored to assess the protective efficacy. Graphs show days post-challenge on the x-axis versus viral loads on the y-axis for VLP (Prime), VLP (Prime-boost) and combined (Prime/Prime-boost). c Right footpad swelling of MAYV challenge groups were monitored to assess protective efficacy. Data represented as mean and SD. P-values were determined by using unpaired T-test. Data represented as mean and SD. P-values were determined by using unpaired T-test, *p < 0.05, **p < 0.01, ****p < 0.0001.