Fig. 1

Development of alphavirus replicon particle vaccine VRP-S-2P against SARS-CoV-2 Omicron variant BA.1. a Schematic diagram of the VEEV-S-2P replicon and VRP construction. The VEEV-S-2P replicon contains two open reading frames (ORF), encoding four non-structural proteins, ns1–ns4, from Venezuelan equine encephalitis virus (VEEV) and the prefusion-stabilized spike protein of SARS-CoV-2 Omicron variant BA.1. The VEEV-S-2P replicon RNA was co-transfected into BHK cells with two helper RNAs expressing the structural proteins of VEEV, resulting in the production of VRP-S-2P. b, c Indirect immunofluorescence and western blot detection of SARS-CoV-2 spike protein followed transfection of VEEV-S-2P and infection of VRP-S-2P at an MOI of 1. Scale bar: 50 μm. d, i The schematic diagram of the VRP-S-2P vaccination in mouse and hamster model. Female Golden Syrian hamsters aged four- to six-weeks and BALB/c aged six- to eight-weeks were immunized with 1×10⁶ FFU VRP-S-2P via intraperitoneal, intramuscular and intranasal routes, respectively. Three immunizations were conducted with two-week intervals. On day 42 post immunization, serum samples were collected. Titers of total RBD-specific IgG (e, j) were measured by ELISA. f, k Neutralizing antibody (PRNT₅₀ titer) against the Omicron variant BA.1 was determined by PRNT. On day 49 post immunization, the immunized mice and hamsters were challenged intranasally with 2 × 10⁴ PFU Omicron variant BA.1. On day 3 post challenge, mice and hamsters were sacrificed. Viral loads in lungs (g, l) and nasal turbinates (h, m) were determined by plaque assay. On day 5 post challenge, hamsters were sacrificed, of which lung tissues were collected. Lung lesions were analyzed by bright field and H&E staining. n Representative lungs from indicated groups of hamsters. Severe pathology in the lungs were depicted by blue arrows. Scale bar = 5 mm. o Representative H&E staining images from indicated groups of hamsters. Arrows for inflammatory cell infiltration (black), eosinophilic secretions (blue), perivascular edema with lymphocyte infiltration or tissue exudate (red), focal lymphoid infiltration around a few bronchi (purple), alveolar hemorrhage (yellow), and lymphocytes infiltration in a sleeve shape and necrotic exfoliated epithelial cells (green) were marked respectively in the images. (Scale bar: 2X = 1 mm, 20X = 100 μm). The above experiments were conducted twice and similar results were obtained. The representative data of one experiment are shown. Data represent the mean ± standard deviation of 4 hamsters at each time point in each group. The asterisks denote statistical differences between the indicated groups. n.s. no statistical difference, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001