key: cord-0751018-cuyk0zzh authors: Hassan, Ahmed O.; Kafai, Natasha M.; Dmitriev, Igor P.; Fox, Julie M.; Smith, Brittany; Harvey, Ian B.; Chen, Rita E.; Winkler, Emma S.; Wessel, Alex W.; Case, James Brett; Kashentseva, Elena; McCune, Broc T.; Bailey, Adam L.; Zhao, Haiyan; VanBlargan, Laura A.; Dai, Yanan; Ma, Meisheng; Adams, Lucas J.; Shrihari, Swathi; Gralinski, Lisa E.; Hou, Yixuan J.; Schaefer, Alexandra; Kim, Arthur S.; Keeler, Shamus P.; Weiskopf, Daniela; Baric, Ralph; Holtzman, Michael J.; Fremont, Daved H.; Curiel, David T.; Diamond, Michael S. title: A single intranasal dose of chimpanzee adenovirus-vectored vaccine confers sterilizing immunity against SARS-CoV-2 infection date: 2020-07-17 journal: bioRxiv DOI: 10.1101/2020.07.16.205088 sha: 72bd0d9ac417b403d0ad136c4f83333bb1553973 doc_id: 751018 cord_uid: cuyk0zzh The Coronavirus Disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of systemic and mucosal IgA and T cell responses, completely prevents SARS-CoV-2 infection in the upper and lower respiratory tracts, and likely confers sterilizing immunity in most animals. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission, and curtailing pandemic spread. To make the vector replication-incompetent and enhance packaging capacity, we replaced the 121 E1A/B genes and introduced a deletion in the E3B gene, respectively (Fig 1A) . To confirm that 122 the S protein was expressed and antigenically intact, we transduced 293T cells and confirmed 123 binding of a panel of 22 neutralizing monoclonal antibodies against the S protein by flow cytometry 124 (Fig 1B) . To assess the immunogenicity of ChAd-SARS-CoV-2-S, groups of 4-week-old BALB/c 126 mice were immunized by intramuscular inoculation with 10 10 virus particles of ChAd-SARS-CoV-127 2-S or ChAd-control. Some mice received a booster dose four weeks later. Serum samples were 128 collected 21 days after primary or booster immunization (Fig 1C) , and IgG responses against 129 purified S and RBD proteins were evaluated by ELISA. Whereas ChAd-SARS-CoV-2-S induced 130 high levels of S-and RBD-specific IgG, low, if any levels were detected in the ChAd-control-131 immunized mice (Fig 1D and S1A) . Serum samples were assayed in vitro for neutralization of 132 infectious SARS-CoV-2 using a focus-reduction neutralization test (FRNT) (Case et al., 2020) . As (Table S1) . Subsequently, quantification of intracellular IFNg and granzyme B expression was determined by 145 flow cytometry. After peptide re-stimulation ex vivo, splenic CD8 + T cells expressed IFNg and both 146 splenic CD4 + and CD8 + T cells expressed granzyme B in mice immunized with ChAd-SARS-CoV-147 2-S but not the ChAd-control vector (Fig 1F-G and S2) . To assess the antigen-specific B cell 148 responses, splenocytes were harvested and subjected to an ELISPOT analysis with S protein. The ChAd-SARS-CoV-2-S vaccine induced S protein-specific IgG antibody-secreting cells in the 150 spleen whereas the control vaccine did not (Fig 1H) . Hu-Ad5 infection (Fig S3A-B) . Five days after Hu-Ad5-hACE2 transduction, mice were challenged via intranasal route 165 with 4 x 10 5 focus-forming units (FFU) of SARS-CoV-2 (Fig 2A) . At 4 days post-infection (dpi), the peak of viral burden in this model (Hassan et al., 2020), mice were euthanized, and lungs, 167 spleen, and heart were harvested for viral burden and cytokine analysis. Notably, there was no 168 detectable infectious virus in the lungs of mice immunized with ChAd-SARS-CoV-2-S as 169 determined by plaque assay, whereas high levels were present in mice vaccinated with ChAd-170 control (Fig 2B) . Consistent with this result, we observed reduced viral RNA levels in the lung, heart, and spleen of ChAd-SARS-CoV-2-S vaccinated animals compared to mice receiving the 172 ChAd-control vector (Fig 2C) . In situ hybridization staining for viral RNA in lungs harvested at 4 173 dpi revealed a substantial decrease of SARS-CoV-2 RNA in pneumocytes of animals immunized 174 with ChAd-SARS-CoV-2-S compared to the ChAd-control (Fig 2D) . A subset of immunized 175 animals was euthanized at 8 dpi, and tissues were harvested for evaluation. At this time point, 176 viral RNA levels again were lower or absent in the lung and spleen of ChAd-SARS-CoV-2-S 177 immunized mice compared to the control ChAd vector (Fig 2C) . Collectively, these data indicate 178 that a single intramuscular immunization with ChAd-SARS-CoV-2-S results in markedly reduced, but not abrogated, SARS-CoV-2 infection in the lungs of challenged mice. We next assessed the effect of the vaccine on lung inflammation and disease. Several 181 proinflammatory cytokines and chemokine mRNA levels were lower in the lung tissues of animals 182 immunized with ChAd-SARS-CoV-2-S compared to ChAd-control including CXCL10, IL1b, IL-6, 183 CCL5, IFNb, and IFNl (Fig 2E) . Moreover, mice immunized with ChAd-control vaccine and 184 challenged with SARS-CoV-2 showed evidence of viral pneumonia characterized by immune cell 185 accumulation in perivascular and alveolar locations, vascular congestion, and interstitial edema. In contrast, animals immunized with ChAd-SARS-CoV-2-S showed a marked attenuation of the 9 inflammatory response in the lung that develops in the ChAd-control-immunized mice (Fig 3) . Thus, immunization with Ch-Ad-SARS-CoV-2 decreases both viral infection and the consequent 189 lung inflammation and injury associated with SARS-CoV-2 infection. We then assessed for improved protection using a prime-boost vaccine regimen. BALB/c 191 mice were immunized via an intramuscular route with ChAd-control or ChAd-SARS-CoV-2-S and 192 received a homologous booster dose four weeks later. At day 29 post-boost, mice were treated 193 with a single dose of anti-Ifnar1 antibody followed by Hu-Ad5-hACE2 and then challenged with 194 SARS-CoV-2 five days later. As expected, the prime-boost regimen protected against SARS- CoV-2 challenge with no infectious virus detected in the lungs (Fig 2F) . Although marked reductions of viral RNA in the lung, spleen, and heart were detected at 4 dpi, residual levels of 197 viral RNA still were present suggesting protection was not complete, even after boosting (Fig 2G) . induced high levels of S-and RBD-specific IgG and IgA (Fig 4B-C) and SARS-CoV-2 neutralizing 207 antibodies (geometric mean titer of 1/1,574) in serum (Fig 4D and S4A ). Serum antibodies from 208 mice immunized with ChAd-SARS-CoV-2-S equivalently neutralized a recombinant, luciferase-209 expressing variant of SARS-CoV-2 encoding a D614G mutation in the S protein ( Fig S4B) ; this 210 finding is important, because many circulating viruses contain this substitution, which is those with neutralizing activity (Fig 4G and S4C ). To assess T cell responses activated via mucosal immunization, mice were vaccinated 2-S vaccine (Fig 4H) . Specifically, a population of IFNg-secreting, antigen-specific 222 CD103 + CD69 + CD8 + T cells in the lung was identified (Fig 4I) which is phenotypically consistent 223 with vaccine-induced resident memory T cells (Takamura, 2017) . In the spleen, we detected 224 antibody-secreting plasma cells producing IgA or IgG against the S protein after intranasal 225 immunization with ChAd-SARS-CoV-2-S (Fig 4J) . Of note, we observed an ~-5-fold higher the extensive inflammation observed in ChAd-control vaccinated animals (Fig 5D) . To determine if sterilizing immunity was achieved with intranasal delivery of ChAd-SARS- CoV-2-S, we measured anti-NP antibodies at 8 dpi and compared them to responses from 5 days and ChAd-SARS-CoV-2-S mice vaccinated by an intramuscular route (Fig 5E and S1D ). Remarkably, none of the mice immunized with ChAd-SARS-CoV-2-S via an intranasal route 251 showed significant increases in anti-NP antibody responses after SARS-CoV-2 infection. Combined with our virological analyses, these data suggest that a single intranasal immunization Tissues were collected at 4 dpi for analysis. Infectious virus in the lung was determined by plaque 430 assay (F) and viral RNA was measured in the lung, spleen and heart using RT-qPCR (G) (n = 6- Virus inoculations were performed under anesthesia that was induced and maintained with 549 ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering. Female BALB/c mice were purchased from The Jackson Laboratory (catalog 000651). Four to five-week-old animals were immunized with 10 10 viral particles (vp) of ChAdV-empty or Hu-AdV5 neutralization assays. One day prior to Hu-AdV5-hACE2 transduction, serum 620 samples were collected from mice immunized intramuscularly with ChAd-Control or ChAd-SARS- CoV-2-S. Sera were heat-inactivated and serially diluted prior to incubation with 10 2 FFU of Hu- Table S1 . SARS-CoV-2 15-mer peptides in S protein Peptide peptide sequence Peptide start Protective efficacy of multiple vaccine 740 platforms against Zika virus challenge in rhesus monkeys Humoral Immunogenicity and Efficacy of a 744 Single Dose of ChAdOx1 MERS Vaccine Candidate in Dromedary Camels A Potently Neutralizing Antibody Protects Mice against 748 SARS-CoV-2 Infection SARS-CoV-2 Vaccines: Status Report The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice A double-inactivated severe acute respiratory 758 syndrome coronavirus vaccine provides incomplete protection in mice and induces increased 759 eosinophilic proinflammatory pulmonary response upon challenge Rational Vaccine Design in the Time of COVID-19 Innovative Mucosal Vaccine Formulations Against Influenza 765 A Virus Infections Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput 769 single-cell sequencing of convalescent patients' B cells Neutralizing antibody and soluble ACE2 inhibition of a 773 replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2. Cell Host and 774 Microbe SARS-CoV-2 infection protects against 778 rechallenge in rhesus macaques Multisystem Inflammatory Syndrome Related to COVID-19 in Previously Healthy 782 Children and Adolescents Covid-19 polyclonal hyperimmune globulin therapy and vaccine development SARS and MERS: recent 789 insights into emerging coronaviruses The Challenges of Vaccine Development against a 792 New Virus during a Pandemic A novel chimpanzee adenovirus vector with low human seroprevalence: 796 improved systems for vector derivation and comparative immunogenicity Tailoring subunit vaccine immunogenicity: maximizing antibody and T cell responses by using 800 combinations of adenovirus, poxvirus and protein-adjuvant vaccines against Plasmodium 801 falciparum MSP1 Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in Neutra Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity Immunogenicity and structures of a rationally 912 designed prefusion MERS-CoV spike antigen Alternative serotype adenovirus vaccine vectors elicit 916 memory T cells with enhanced anamnestic capacity compared to Ad5 vectors Cross-neutralization of SARS-CoV-2 by a human 921 monoclonal SARS-CoV antibody Prime-boost immunization with adenoviral and modified 925 vaccinia virus Ankara vectors enhances the durability and polyfunctionality of protective malaria 926 CD8+ T-cell responses Creation of a panel of vectors based on ape adenovirus isolates Neutralizing Monoclonal Antibodies against Hepatitis C Virus 933 E2 Protein Bind Discontinuous Epitopes and Inhibit Infection at a Postattachment Step Blocking monoclonal antibodies specific 938 for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo 939 hydrodynamic transfection How advances in immunology provide insight into improving 941 vaccine efficacy Persistence in Temporary Lung Niches: A Survival Strategy of Lung-944 Resident Memory CD8(+) T Cells Immunization with modified vaccinia virus Ankara-based 948 recombinant vaccine against severe acute respiratory syndrome is associated with enhanced 949 hepatitis in ferrets Autopsy Findings and Venous 953 Thromboembolism in Patients With COVID-19: A Prospective Cohort Study Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation DNA vaccine protection against SARS-CoV-2 in 965 rhesus macaques A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-969 Current prospects and future challenges for nasal vaccine delivery Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, 976 China: a retrospective cohort study A pneumonia outbreak associated with a new coronavirus of probable bat 980 origin Safety, tolerability, and immunogenicity of a recombinant adenovirus type-984 COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human 985 trial Rapid isolation and profiling of a diverse panel of 989 human monoclonal antibodies targeting the SARS-CoV-2 spike protein Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12.