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...

Utilization of antibodies to deliver highly potent cytotoxic agents to corresponding antigen-overexpressed tumor cells is a clinically validated therapeutic strategy. Ofatumumab (OFA, trade name Arzerra) is a fully human CD20-specific antibody that is active against CD20-positive B-cell lymphoma/chronic lymphocytic leukemia cells. In order to further enhance the anticancer effect of OFA, anti-CD20 OFA has been conjugated with highly cytotoxic monomethyl auristatin E (MMAE) through a cathepsin-B-cleavable valine-citrulline (vc) dipeptide linkage to form OFA-vcMMAE and the anti-tumor activity of OFA-vcMMAE against CD20-positive B lymphoma cells are then evaluated in vitro and in vivo. As a result, conjugation of OFA with MMAE has kept the initial effector functional activities of OFA such as binding affinity, complement-dependent cytotoxicity (CDC) as well as antibody-dependent cell-mediated cytotoxicity (ADCC). In addition, the conjugation of MMAE significantly improved the cytotoxic activity of OFA against CD20-positive cells (i.e., Raji, Daudi and WIL2-S cells) but not against CD20-negative K562 cells. On the other hand, OFA-vcMMAE was modulated from the CD20-positive cell surface and then entered the lysosomes by receptor-mediated endocytosis, underwent proteolytic degradation and released active drug MMAE to induce apoptotic cell death through a caspase-3-like protease-dependent pathway. Surprisingly, OFA-vcMMAE completely inhibited the growth of CD20-positive Daudi and Ramos lymphoma xenografts in vivo, and exhibited greater anti-tumor activity than unconjugated OFA, suggesting that the anti-tumor activity of anti-CD20 antibody can be enhanced by conjugation with MMAE. In the near future, this new approach might be used as a clinical treatment of CD20-positive B lymphoid malignancies.

In our previous study, we have demonstrated in the context of WNV-ΔNS1 vaccine (a replication-defective West Nile virus (WNV) lacking NS1) that the NS1 trans-complementation system may offer a promising platform for the development of safe and efficient flavivirus vaccines only requiring one dose. Here, we produced high titer (10 7 IU/ml) replication-defective Japanese encephalitis virus (JEV) with NS1 deletion (JEV-ΔNS1) in the BHK-21 cell line stably expressing NS1 (BHK NS1) using the same strategy. JEV-ΔNS1 appeared safe with a remarkable genetic stability and high degrees of attenuation of in vivo neuroinvasiveness and neurovirulence. Meanwhile, it was demonstrated to be highly immunogenic in mice after a single dose, providing similar degrees of protection to SA14-14-2 vaccine (a most widely used live attenuated JEV vaccine), with healthy condition, undetectable viremia and gradually rising body weight. Importantly, we also found JEV-ΔNS1 induced robust cross-protective immune responses against the challenge of heterologous West Nile virus (WNV), another important member in the same JEV serocomplex, accounting for up to 80% survival rate following a single dose of immunization relative to mock-vaccinated mice. These results not only support the identification of the NS1-deleted flavivirus vaccines with a satisfied balance between safety and efficacy, but also demonstrate the potential of the JEV-ΔNS1 as an alternative vaccine candidate against both JEV and WNV challenge.