![]() ![]() To evaluate the immunogenicity of VRP-S-2P, different inoculation routes were performed in the mouse model which had been demonstrated to be sensitive to SARS-CoV-2 Omicron variant infection. The asterisks denote statistical differences between the indicated groups. Data represent the mean ± standard deviation of 4 hamsters at each time point in each group. The representative data of one experiment are shown. The above experiments were conducted twice and similar results were obtained. 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. o Representative H&E staining images from indicated groups of hamsters. Severe pathology in the lungs were depicted by blue arrows. n Representative lungs from indicated groups of hamsters. Lung lesions were analyzed by bright field and H&E staining. On day 5 post challenge, hamsters were sacrificed, of which lung tissues were collected. Viral loads in lungs ( g, l) and nasal turbinates ( h, m) were determined by plaque assay. On day 3 post challenge, mice and hamsters were sacrificed. On day 49 post immunization, the immunized mice and hamsters were challenged intranasally with 2 × 10 4 PFU Omicron variant BA.1. f, k Neutralizing antibody (PRNT 50 titer) against the Omicron variant BA.1 was determined by PRNT. Titers of total RBD-specific IgG ( e, j) were measured by ELISA. On day 42 post immunization, serum samples were collected. Three immunizations were conducted with two-week intervals. Female Golden Syrian hamsters aged four- to six-weeks and BALB/c aged six- to eight-weeks were immunized with 1×10 6 FFU VRP-S-2P via intraperitoneal, intramuscular and intranasal routes, respectively. d, i The schematic diagram of the VRP-S-2P vaccination in mouse and hamster model. 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. 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. 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. a Schematic diagram of the VEEV-S-2P replicon and VRP construction. 2ĭevelopment of alphavirus replicon particle vaccine VRP-S-2P against SARS-CoV-2 Omicron variant BA.1. ![]() 2 The alphavirus VRP system has been extensively used in cancer therapy and vaccine development with well-demonstrated safety and tolerability in numerous clinical trials. ![]() The replicon could be encapsulated into single-round infectious alphavirus replicon particles (VRPs) by in trans supplement of the viral structural proteins and thereafter be delivered into cells by VRP infection. Additionally, the replicable saRNA could activate an innate immune response, which has the potential to provide an adjuvant effect on vaccine potency. Compared with the conventional non-replicating mRNA, the alphavirus-derived self-amplifying replicon RNA (saRNA) encodes the viral replicase as well as a gene of interest (GOI), which enables the RNA replication and GOI expression in the cytoplasm 2 and circumvents the risk of genomic integration, thus enhances the safety profile of the saRNA-based vaccines. VEEV is a positive-sense, single-stranded RNA virus that belongs to the genus alphavirus of the family Togaviridae. In this study, we generated a SARS-CoV-2 Omicron variant vaccine using Venezuelan equine encephalitis virus (VEEV) replicon to encode the prefusion-stabilized spike protein of the Omicron variant BA.1. ![]()
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