A mouse model for SARS-CoV-2 infection by exogenous delivery of hACE2 using alphavirus replicon particles

Abstract

Dear Editor, Since the outbreak of a novel coronavirus disease (COVID-19) in late 2019, it has spread rapidly and developed into a global pandemic. As of August 12, 2020, more than 215 countries and territories around the world have reported more than 20.5 million confirmed COVID-19 cases with over 745,693 deaths (https://www. worldometers.info/coronavirus/#countries). Such harsh conditions urged scientists across the world to gear up to develop vaccines and antiviral drugs against COVID-19, which also lead to massive requirement for experimental animals. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of COVID-19. It has been demonstrated that SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as cellular receptor for entry into target cells. Mouse model is the most commonly used animal model for studying human diseases. However, SARS-CoV-2 fails to invade and replicate in this traditional animal model due to the structural differences in mouse ACE2 (mACE2) compared with human ACE2 (hACE2), 1 which has become the major hurdle for COVID-19 study. Currently, several strategies have been developed to overcome this receptor incompatibility by: (i) generating transgenic mice bearing hACE2 receptor, 2-4 (ii) establishing adenovirus hACE2 mouse model with recombinant adenovirus expressing hACE2, 5 and (iii) adapting the SARS-CoV-2 by serial passages in the respiratory tract of mice. 6-8 In this study, we used an alternative strategy to generate a SARS-CoV-2-sensitive mouse model by exogenous delivery of hACE2 with Venezuelan equine encephalitis replicon particles (VEEV-VRP-hACE2) (Supplementary information, Fig. S1a). VEEV is a positive sense, single-stranded RNA virus which belongs to the genus Alphavirus, family Togaviridae. Alphavirus replicon particles (VRPs), including VEEV-VRPs, represent efficient vectors for gene delivery and have been applied to studies of vaccine development, gene therapy and cell transduction. They contain self-replicating RNAencoding viral replicase proteins (nsP1-nsP4) and express the gene of interest in place of viral structural protein genes. 9 By providing viral structural proteins in trans, the replicon RNA is packaged into VEEV-VRPs for in vitro and in vivo gene delivery. 10 Due to their intrinsic biological properties, VEEV-VRPs offer several advantages with a broad range of susceptible host cells, high expression level of cytoplasmic proteins and easy manipulation of recombinant RNA molecules using cDNA clones. 10,11 Here, Venezuelan equine encephalitis virus (VEEV) replicon expressing hACE2 with a C-terminal Stag was packaged into VRPs using the helper RNAs encoding VEEV capsid and envelope proteins to produce VEEV-VRP-hACE2 (Supplementary information, Fig. S1). MLE-12 cells (mouse lung type II epithelial cell line) were used to evaluate the availability of VEEV-VRP-hACE2 for SARS-CoV-2-sensitive cells establishment. After confirming hACE2 expression in MLE-12 cells transduced with VEEV-VRP-hACE2 (VRP-hACE2) through indirect immunofluoresc...

ACE2-targeting monoclonal antibody as potent and broad-spectrum coronavirus blocker

“…This model was generated by exogenous delivery of hACE2 with Venezuelan equine encephalitis replicon particles, VEEV-VRP-hACE2. 37 It was a non-lethal infection model that was suitable for measuring viral RNA loads and histological pathology of lungs. After hACE2 was intranasally delivered by VEEV, the antibodies were administered intraperitoneally at 10 mg/kg, and mice were challenged intranasally with 10 5 PFU of live SARS-CoV-2 12 h later.…”

“…A mouse model recently established by VEEV-VRP delivery of ACE2 for SARS-CoV-2 infection 37 was used to evaluate the efficacy of 3E8 in vivo. Four groups of 6–8-week-old female BALB/c mice ( n = 5 per group) were first intranasally infected with 10 6 FFU VRP-ACE2 per mouse in a total volume of 80 μl after anesthetization with Avertin (250 mg/kg).…”

Immune responses to SARS-CoV-2 infection in Humans and ACE2 humanized mice

“…Contrast to the lymphocytes increasing in the lung, the number of B cells, CD4+ T cells, CD8+ T cells and monocytes in the peripheral blood markedly decreased in K18-hACE2 mice at 5 d.p.i., accompanied by an increased neutrophil-to-lymphocyte ratio, similar to COVID-19 patients 78 . As evidenced by Emma S. Winkler, Venezuelan equine encephalitis replicon particles (VEEV-VRP-hACE2)-transduced, SARS-CoV-2-infected mice had a higher neutrophil-to-lymphocyte ratio in the peripheral blood at 2 dpi compared with control mice 79 . All these studies focused on lymphocyte responses to SARS-CoV-2 byanalyzing bulk T cells rather than SARS-CoV-2 specific T cell responses.…”

ACE2-Targeting Monoclonal Antibody as Potent and Broad-Spectrum Coronavirus Blocker

“…More importantly, the neutralizing ability of 3E8 was validated in a mouse model of COVID-19. This model was generated by exogenous delivery of hACE2 with Venezuelan equine encephalitis replicon particles, VEEV-VRP-hACE2, and was published previously ( 30 ). After VEEV-VRP-hACE2 transfection and antibody application, mice were infected with 10 5 PFU of live SARS-CoV-2 via intranasal route, and then viral RNA loads and tissue damages in lungs were examined 3 days post infection.…”