key: cord-0914621-dawejxvd authors: Case, James Brett; Rothlauf, Paul W.; Chen, Rita E.; Kafai, Natasha M.; Fox, Julie M.; Smith, Brittany; Shrihari, Swathi; McCune, Broc T.; Harvey, Ian B.; Keeler, Shamus P.; Bloyet, Louis-Marie; Zhao, Haiyan; Ma, Meisheng; Adams, Lucas J.; Winkler, Emma S.; Holtzman, Michael J.; Fremont, Daved H.; Whelan, Sean P.J.; Diamond, Michael S. title: Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis in mice date: 2020-07-30 journal: Cell Host Microbe DOI: 10.1016/j.chom.2020.07.018 sha: 30fa8caead381bdbf8e5f044ebd1ad6f7d3225ba doc_id: 914621 cord_uid: dawejxvd SUMMARY Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and an effective vaccine is critical to mitigate coronavirus-induced disease 2019 (COVID-19). Previously, we developed a replication-competent vesicular stomatitis virus (VSV) expressing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Here, we show that vaccination with VSV-eGFP-SARS-CoV-2 generates neutralizing immune responses and protects mice from SARS-CoV-2. Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high antibody titers that neutralize SARS-CoV-2 and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung, indicating protection against pneumonia. Passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals also protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense, To examine the immune response to VSV-eGFP-SARS-CoV-2, we immunized four- week-old BALB/c mice with 10 6 plaque-forming units (PFU) of VSV-eGFP-SARS-CoV-2 or a 95 control, VSV-eGFP ( Fig 1A) . As murine ACE2 does not serve as a receptor for SARS-CoV-2, 96 we spiked our preparation of VSV-eGFP-SARS-CoV-2 with trace amounts of VSV G to permit a 97 single round of infection, an approach used previously for SARS-CoV (Kapadia et al., 2008) 98 (Fig S1) . At 28 days post-priming, one cohort of animals was boosted with the homologous 99 vaccine. Serum was isolated from all animals at three weeks post priming or boosting, and IgG 100 titers against recombinant SARS-CoV-2 S protein or the RBD were determined by ELISA ( Fig 101 1B-C) . Immunization with VSV-eGFP-SARS-CoV-2 induced high levels of anti-S and anti-RBD-102 specific IgG compared to control VSV-eGFP with reciprocal median serum endpoint titers of 3.2 103 x 10 5 and 2.7 x 10 6 (anti-S) and 1.1 x 10 4 and 1.4 x 10 5 (anti-RBD) for one and two doses of 104 vaccine, respectively. We measured neutralizing antibody titers against SARS-CoV-2 after priming or boosting 106 using a focus-reduction neutralization test (Case et al., 2020) . Immunization with a single or 107 two-dose regimen of VSV-eGFP-SARS-CoV-2 induced neutralizing antibodies (median titers of 108 1/59 and 1/5206, respectively) whereas the control VSV-eGFP vaccine did not (Fig 1D) . Boosting was effective and resulted in a 90-fold increase in neutralizing activity after the second 110 dose of VSV-eGFP-SARS-CoV-2. Collectively, these data suggest that VSV-eGFP-SARS-CoV-111 6 2 is immunogenic and elicits high titers of antibodies that neutralize infection and target the RBD 112 of the SARS-CoV-2 S protein. Because VSV-eGFP-SARS-CoV-2 might not enter efficiently into cells of conventional 114 BALB/c mice lacking the human ACE2 (hACE2) receptor, we confirmed immunogenicity in K18-115 hACE2 transgenic C57BL/6 mice, in which hACE2 expression is driven by an epithelial cell 116 promoter (McCray et al., 2007) . We immunized four-week-old K18-hACE2 transgenic mice with 117 10 6 PFU of VSV-eGFP-SARS-CoV-2 or VSV-eGFP control. Serum was isolated at three weeks 118 post-priming, and IgG titers against recombinant SARS-CoV-2 RBD were measured by ELISA. We detected robust IgG responses against RBD in VSV-eGFP-SARS-CoV-2 but not VSV-eGFP 120 vaccinated mice (Fig 1E) . Immunoglobulin subclass analysis indicated substantial class-121 switching occurred, as high levels of IgG2b and IgG2c against RBD were detected (Fig 1E) . Finally, we detected neutralizing antibodies against SARS-CoV-2 (median titer of 1/325) three 123 weeks after immunizing K18-hACE2 transgenic mice with a single dose of VSV-eGFP-SARS- CoV-2 but not VSV-eGFP (Fig 1F) . x 10 5 PFU of SARS-CoV-2 (strain 2019 n-CoV/USA_WA1/2020) to evaluate vaccine protection 133 (Fig 1A) . We subsequently measured viral yield both by plaque forming and RT-qPCR assays. At day 4 post-infection (dpi) infectious virus was not recovered from lungs of mice vaccinated 135 either with one or two doses of VSV-eGFP-SARS-CoV-2 (Fig 2A) . For mice receiving only one 136 dose of VSV-eGFP-SARS-CoV-2 vaccine, we observed a trend towards decreased levels of 7 viral RNA in the lung, spleen, and heart at 4 dpi and in the lung and spleen at 8 dpi compared to 138 the control VSV-eGFP vaccinated mice (Fig 2B-E) . The low levels of SARS-CoV-2 infection in 139 the heart, which were observed previously in this model (Hassan et al., 2020) , may be due to 140 spread of the AdV-hACE2 from venous circulation in the lung. Mice that received two doses of 141 VSV-eGFP-SARS-CoV-2 had significantly lower levels of viral RNA in most tissues examined 142 compared to control VSV-eGFP vaccinated mice (Fig 2B-E) . Consistent with our viral RNA 143 measurements, we observed less SARS-CoV-2 RNA by in situ hybridization in lung tissues of 144 VSV-eGFP-SARS-CoV-2 immunized mice at 4 dpi (Fig 2F) . Collectively, these data support that immunizations. Ten-week-old female BALB/c mice were administered anti-Ifnar1 mAb and AdV-176 hACE2 as described above to render animals susceptible to SARS-CoV-2. Five days later, 100 177 μL of pooled immune or control sera was administered by intraperitoneal injection. One day 178 later, mice were inoculated with 3 x 10 5 PFU of SARS-CoV-2 via the intranasal route (Fig 4A) . Passive transfer of sera from animals vaccinated with VSV-eGFP-SARS-CoV-2 protected 180 against SARS-CoV-2 infection compared to sera from the VSV-eGFP-immunized mice. At 4 dpi, 181 lungs from animals treated with VSV-eGFP-SARS-CoV-2 immune sera from prime-only and 182 boosted animals showed substantially reduced infectious virus burden (Fig 4B) . Although not as 183 striking, significant decreases in viral RNA levels also were observed in the lung and spleen of 184 animals receiving VSV-eGFP-SARS-CoV-2 boosted sera compared to the VSV-eGFP sera ( Fig 185 4C-D) . Possibly, some of the viral RNA in lung tissue homogenates after passive transfer may 186 represent neutralized virus within cells that has not yet been cleared. Viral RNA levels in the 187 heart of animals given sera from VSV-eGFP-SARS-CoV-2 boosted mice trended toward, but did 9 not reach, statistical significance (Fig 4E) . No effect was observed in the nasal washes of any 189 treated group (Fig 4F) , consistent with the results from our vaccinated and challenged animals 190 (Fig 2E) . To determine the effect of the passive transfer of sera on SARS-CoV-2-mediated 192 inflammation, we assessed the induction of several cytokines in the lung at 4 dpi (Fig 4G) . Chi, X., Yan, R., Zhang, J., Zhang, G., Zhang, Y., Hao, M., Zhang, Z., Fan, P., Dong, Y., Yang, Ecological associations of vesicular stomatitis virus in rural Central America 616 and Panama Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in 620 the lungs Recombinant vesicular stomatitis virus vector 623 vaccines for WHO blueprint priority pathogens Eukaryotic transient-expression 627 system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA 628 polymerase A single dose of a vesicular stomatitis virus-based 632 influenza vaccine confers rapid protection against H5 viruses from different clades Properties of replication-competent vesicular 640 stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses Vesicular stomatitis virus-645 based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates Development of a new vaccine for the 650 prevention of Lassa fever Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals Pathology and pathogenesis of severe acute respiratory 657 syndrome A SARS-CoV-2 Infection Model in Mice 661 Demonstrates Protection by Neutralizing Antibodies Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring 666 vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!) Efficacy and effectiveness 670 of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the 671 Guinea ring vaccination cluster-randomised trial Clinical features of patients infected with 2019 novel coronavirus in Wuhan The effect of dose on the safety and 679 immunogenicity of the VSV Ebola candidate vaccine: a randomised double-blind, placebo-680 controlled phase 1/2 trial Syrian hamsters as a small animal model for 684 SARS-CoV-2 infection and countermeasure development Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human 689 Angiotensin-Converting Enzyme 2. Cell Clinical and serological response to 692 laboratory-acquired human infection by Indiana type vesicular stomatitis virus (VSV) Live attenuated recombinant vaccine 697 protects nonhuman primates against Ebola and Marburg viruses SARS vaccine based on a replication-700 defective recombinant vesicular stomatitis virus is more potent than one based on a replication-701 competent vector Phase 2 Placebo-Controlled Trial of Two Vaccines to 705 Prevent Ebola in Liberia Functional assessment of cell entry and receptor 708 usage for SARS-CoV-2 and other lineage B betacoronaviruses A unique strategy for mRNA cap methylation used 711 by vesicular stomatitis virus Vesicular stomatitis virus: re-714 inventing the bullet Developing Covid-19 Vaccines at 717 Pandemic Speed mRNA cap methylation influences pathogenesis of vesicular 721 stomatitis virus in vivo Vesicular 724 stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host Lethal infection of K18-hACE2 mice infected 729 with severe acute respiratory syndrome coronavirus Recombinant vesicular stomatitis virus 733 vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, 738 circumvent humoral immunity, and enhance oncolytic potency Enhancing dengue virus maturation using a stable furin over-expressing cell line Cross-neutralization of SARS-CoV-2 by a human 746 monoclonal SARS-CoV antibody Comparative neurovirulence of 749 selected vesicular stomatitis virus temperature-sensitive mutants of complementation groups II 750 and III Attenuated vesicular 753 stomatitis viruses as vaccine vectors Isolation of potent SARS-CoV-2 neutralizing antibodies and 757 protection from disease in a small animal model Heterologous prime/boost immunization 761 of rhesus monkeys by using diverse poxvirus vectors Blocking monoclonal antibodies 765 specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo 766 hydrodynamic transfection CoV-2 in golden hamsters A recombinant vesicular stomatitis virus 773 bearing a lethal mutation in the glycoprotein gene uncovers a second site suppressor that 774 restores fusion A Mouse Model of SARS-CoV-2 Infection and Pathogenesis Chimeric Viruses with Vesicular Stomatitis 782 Virus: Actions in the Brain Recoding of the vesicular stomatitis virus L gene by computer-aided design provides a live, 786 attenuated vaccine candidate Gene rearrangement attenuates 789 expression and lethality of a nonsegmented negative strand RNA virus Efficient recovery of infectious 793 vesicular stomatitis virus entirely from cDNA clones Identification of a minimal size requirement 796 for termination of vesicular stomatitis virus mRNA: implications for the mechanism of 797 transcription Lassa-800 vesicular stomatitis chimeric virus safely destroys brain tumors DNA vaccine protection CoV-2 in rhesus macaques Safety, tolerability, and immunogenicity of a recombinant adenovirus 808 type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-809 human trial CoV-2 vaccine induces high-titer neutralizing antibodies • Infectious SARS-CoV-2 is undetectable in the lung of vaccinated mice post-challenge • SARS-CoV-2-induced lung inflammation and pathology is decreased in vaccinated mice • Transfer of vaccine-derived immune sera to naïve mice protects against SARS report the efficacy of a replicating VSV-based SARS-CoV-2 vaccine. Immunized hACE2-expressing mice challenged with SARS-CoV-2 are protected against lung infection, inflammation, and pneumonia. Neutralizing antibodies are a correlate of this protection, as passive transfer of vaccine-derived immune sera protects naïve mice from subsequent SARS-CoV-2 challenge