Development of a Broadly Accessible Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Platform

Abstract

Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations, causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this work, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate the utility of the VRP 3526 platform in the generation of recombinant proteins, production of virus-like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under biosafety level 2 (BSL2) conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of laboratories around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to that of previously attenuated VRP approaches. Taking these results together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens.IMPORTANCE While VEE virus replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high-containment-level facilities for production and packaging. In this work, we utilize an attenuated vaccine strain capable of use at lower biocontainment level but retaining the capacity of the wild-type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence.

Keywords: VEE replicon; VRP; aged; coronavirus; heterologous; norovirus; vaccine.

FIG 1

Characterization of VRP 3526 platform. (A) Schematic of VRP 3526 platform showing replicon gene with reporter gene in pVR21 plasmid. Arrows indicate the start of 26S subgenomic promoter. (B) Titers of S protein vaccines from V3000, V3526, and V3014 coats determined on BHK cells by an IFA assay. **, P < 0.01; ***, P < 0.001 (Student's t test). (C) Western blot from independent experiments showing expression of SARS-CoV Spike protein from V3526S and V3000S vaccines in Vero cells. The lower panel shows actin.

FIG 2

Morphology, ligand binding, and antigenicity of Norwalk virus VLPs produced from V3014 or V3526 VRPs. (A and B) Electron micrograph of Norwalk virus VLPs produced from V3014 (A) or V3526 (B) VRPs. (C) Carbohydrate ligand binding of Norwalk virus VLPs from V3526 (blue) and V3014 (gray). (D) Mouse anti-Norwalk virus polyclonal serum binding to Norwalk virus VLPs. Plotted markers represent the means and standard deviations.

FIG 3

V3526S protects young mice from lethal SARS-CoV disease induced by homologous challenge. (A and B) Percent weight loss (A) and survival curve (B) of young mice immunized with S protein-based vaccines from V3000S (black; n = 11), V3526S (blue; n = 11), V3014S (gray; n = 11), or control (VRP-HA; dashed line; n = 11) challenged with 10⁵ PFU of SARS-CoV MA15. (C) Lung virus titers on 2 days postinfection determined by plaque assay on Vero cells (n = 5 for VRP, n = 3 for control). The dashed line represents the limit of detection for plaque assay. Error bars, SEM; ND, none detected; ***, P < 0.001 (Student's t test). (D) Representative H&E-stained lung sections harvested 4 days postinfection from the indicated vaccine groups showing lungs, alveoli, and airway vasculature at lower (40×) and higher (400×) magnification. Note the presence of hyaline membranes (yellow arrow) in animals vaccinated with VRP-HA, indicating end-stage lung disease, which is absent from V3000S and V3526S vaccine groups. (E) Scoring of clinical disease in H&E-stained lung sections harvested 4 days postinfection with V3000S (black), V3526S (blue), V3014S (gray), or control (VRP-HA; white) for airway disease, vascular cuffing, pneumonia, and diffuse alveolar damage (DAD).

FIG 4

V3526S protects young mice from SARS-CoV disease induced by heterologous challenge. (A) Percent weight loss of young mice immunized with S protein-based vaccines from V3000S (black; n = 11), V3526S (blue; n = 15), or mock (gray; n = 7) immunization and challenge with 10⁵ PFU of rMA15-GD03. (B) Lung virus titers at 2 days postinfection determined by plaque assay on Vero cells with V3000S (black; n = 3), V3526S (blue; n = 4), or mock (gray; n = 3) immunization. Error bars indicate standard errors of the means (SEM). ***, P < 0.001 (Student's t test). (C) Representative H&E-stained lung sections from V3000S- or V3526S-vaccinated mice harvested 4 days postinfection showing lungs, alveoli, and airway vasculature at lower (40×) and higher (400×) magnification. (D) Scoring of clinical disease in H&E-stained lung sections harvested 4 days postinfection from the indicated V3000S (black)- or V3526S (blue)-vaccinated mice for airway disease, vascular cuffing, pneumonia, and diffuse alveolar damage (DAD). Error bars indicate standard deviations (SD).

FIG 5

V3526S protects aged mice from lethal SARS-CoV disease. (A to C) Percent weight loss (A) and survival curve (B) of aged mice immunized with S protein-based vaccines from V3000S (black; n = 10), V3526S (blue; n = 12), V3014S (gray; n = 6), or control (VRP HA; dashed; n = 7) challenge with 10⁵ PFU of SARS-CoV MA15. (C) Lung virus titers at 2 days postinfection and 4 days postinfection, determined by plaque assay on Vero cells. V3000S (black; n = 6 [day 2], 3 [day 4]), V3526S (blue; n = 5 [day 2], 3 [day 4]), V3014S (gray, n = 3), and control (VRP HA; white; n = 4 [day 2], 3 [day 4]) are shown. Error bars indicate SEM. *, P < 0.05; ***, P < 0.001 (Student's t test). (D) Representative H&E-stained lung sections harvested 4 days postinfection from the indicated vaccine groups showing lungs, alveoli, and airway vasculature at lower (40×) and higher (400×) magnification. Note clean airways in V3526S and V3000S groups, as indicated by green arrows, and denuded airways in V3014S (green arrow). Note the massive inflammatory infiltrates (yellow arrows) in V3014S and VRP-HA groups, which are reduced in V3526S and V3000S groups.

FIG 6

V3526S induces high antibody titers that neutralize SARS-CoV. (A) ELISA results showing IgG titers to S protein, elicited in young mice following vaccination with V3000S (black), V3526S (blue), V3014S (gray), or control VRP-HA (white). (B) Th1/Th2 skewing as measured by IgG_2A/IgG1 ratio in young mice. (C) ELISA results for aged mice following vaccination by indicated vaccine groups. (D) Th1/Th2 skew in aged mice as measured by IgG_2A/IgG1 ratios. (E and F) Neutralization potential (SARS-CoV) of antibodies elicited by indicated vaccine group, i.e., V3000S (black), V3526S (blue), V3014S (gray), or control VRP-HA (dashed), in young mice (E) and aged mice (F), as measured by PRNT₅₀ assay. ***, P < 0.001 (Student's t test) relative to V3000S and V3526S.