https://doi.org/10.1038/s41586-020-2607-z Accelerated Article Preview Single-shot Ad26 vaccine protects against W SARS-CoV-2 in rhesus macaques E VI Received: 20 June 2020 Noe B. Mercado, Roland Zahn, Frank Wegmann, Carolin Loos, Abishek Chandrashekar, Jingyou Yu, Jinyan Liu, Lauren Peter, Katherine McMahan, Lisa H. Tostanoski,E Xuan He, Accepted: 24 July 2020 David R. Martinez, Lucy Rutten, Rinke Bos, Danielle van Manen, Jort Vellinga, Jerome Custers, Accelerated Article Preview Published Johannes P. Langedijk, Ted Kwaks, Mark J. G. Bakkers, DavidR Zuijdgeest, online 30 July 2020 Sietske K. Rosendahl Huber, Caroline Atyeo, Stephanie Fischinger, John S. Burke, Jared Feldman, Blake M. Hauser, Timothy M. Caradonna,P Esther A. Bondzie, Gabriel Dagotto, Cite this article as: Mercado, N. B. et al. Makda S. Gebre, Emily Hoffman, Catherine Jacob-Dolan, Marinela Kirilova, Zhenfeng Li, Single-shot Ad26 vaccine protects against Zijin Lin, Shant H. Mahrokhian, Lori F. Maxfield, Felix Nampanya, Ramya Nityanandam, SARS-CoV-2 in rhesus macaques. Nature Joseph P. Nkolola, Shivani Patel, John D. Ventura,E Kaylee Verrington, Huahua Wan, https://doi.org/10.1038/s41586-020-2607-z Laurent Pessaint, Alex Van Ry, Kelvin Blade, Amanda Strasbaugh, Mehtap Cabus, (2020). Renita Brown, Anthony Cook, Serge Zouantchangadou,L Elyse Teow, Hanne Andersen, Mark G. Lewis, Yongfei Cai, Bing Chen, Aaron G. Schmidt, R. Keith Reeves, Ralph S. Baric, Douglas A. Lauffenburger, Galit Alter,C Paul Stoffels, Mathai Mammen, Johan Van Hoof, Hanneke Schuitemaker &T DanI H. Barouch This is a PDF fle of a peer-reviewed paper that has been accepted for publication. Although unedited,R the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. TheA text and fgures will undergo copyediting and a proof review before the paper is published in its fnal form. Please note that during the production process errorsD may be discovered which could afect the content, and all legal disclaimers Eapply. T A R E L E C C A Nature | www.nature.com Article Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques W 1,10 2,10 2,10 3,4,10 https://doi.org/10.1038/s41586-020-2607-z Noe B. Mercado , Roland Zahn , Frank Wegmann , Carolin Loos , Abishek 1,10 1,10 1,10 1,10 E1,10 Chandrashekar , Jingyou Yu , Jinyan Liu , Lauren Peter , Katherine McMahan , Received: 20 June 2020 Lisa H. Tostanoski1,10, Xuan He1,10, David R. Martinez5,10, Lucy Rutten2, Rinke Bos2I, Accepted: 24 July 2020 Danielle van Manen2, Jort Vellinga2, Jerome Custers2, Johannes P. Langedijk2, Ted Kwaks2, 2 2 2 V 3,6 Mark J. G. Bakkers , David Zuijdgeest , Sietske K. Rosendahl Huber , Caroline Atyeo , Published online: 30 July 2020 Stephanie Fischinger3,6, John S. Burke3, Jared Feldman3,6, Blake M. Hauser3,6, Timothy M. Caradonna3,6, Esther A. Bondzie1, Gabriel Dagotto1,6, MakdaE S. Gebre1,6, Emily Hoffman1, Catherine Jacob-Dolan1,6, Marinela Kirilova1, Zhenfeng Li1, Zijin Lin1, Shant H. Mahrokhian1, Lori F. Maxfield1, Felix Nampanya1, RamyaR Nityanandam1, Joseph P. Nkolola1, Shivani Patel1, John D. Ventura1, Kaylee Verrington1, Huahua Wan1, Laurent Pessaint7, Alex Van Ry7, Kelvin Blade7, AmandaP Strasbaugh7, Mehtap Cabus7, Renita Brown7, Anthony Cook7, Serge Zouantchangadou7, Elyse Teow7, Hanne Andersen7, Mark G. Lewis7, Yongfei Cai8, Bing Chen8,9, Aaron G. Schmidt3,6,9, R. Keith Reeves1, Ralph S. Baric5, Douglas A. Lauffenburger4, GalitE Alter3,9, Paul Stoffels2, Mathai Mammen2, Johan Van Hoof2, Hanneke SchuitemakerL2,11 & Dan H. Barouch 1,3,6,9,11 ✉ A safe and efective vaccine for severeC acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be requiredI to end the coronavirus disease 2019 (COVID-19) pandemic1–8. For global deploymentT and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efcacy of a single doseR of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in nonhuman primates. Fifty-two rhesus macaques wereA immunized with Ad26 vectors encoding S variants or sham control and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26D vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs following ESARS-CoV-2 challenge. Vaccine-elicited neutralizing antibody titres correlated with protective efcacy, suggesting an immune correlate of protection. These data T demonstrate robust single-shot vaccine protection against SARS-CoV-2 in nonhuman A primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is R currently being evaluated in clinical trials. The rapid expansion of the COVID-19 pandemic has made the development mutations: (i) tissue plasminogen activator (tPA) leader sequence with of a SARS-CoV-2 vaccineE a global health and economic priority. Adenovirus full-length S (tPA.S)12, (ii) tPA leader sequence with full-length S with serotype 26 (Ad26) vectors11 encoding viral antigens have been shown to mutation of the furin cleavage site and two proline stabilizing mutations induce robust humoralL and cellular immune responses to various patho- (tPA.S.PP)13–15, (iii) wildtype leader sequence with native full-length S gens in both nonhuman primates and humans. In this study, we developed (S), (iv) wildtype leader sequence with S with deletion of the cytoplas- a series of Ad26E vectors encoding different variants of the SARS-CoV-2 spike mic tail (S.dCT)16, (v) tandem tPA and wildtype leader sequences with (S) protein and evaluated their immunogenicity and protective efficacy full-length S (tPA.WT.S)12, (vi) wildtype leader sequence with S with againstC SARS-CoV-2 challenge in rhesus macaques. deletion of the transmembrane region and cytoplasmic tail reflecting the soluble ectodomain, with mutation of the furin cleavage site, proline C stabilizing mutations, and a foldon trimerization domain (S.dTM.PP)15, Generation and Immunogenicity of Ad26 Vaccine and (vii) wildtype leader sequence with full-length S with mutation of A Candidates the furin cleavage site and proline stabilizing mutations (S.PP) (Fig. 1a). We produced seven Ad26 vectors expressing SARS-CoV-2 S variants that Western blot analyses confirmed S expression in cell lysates from all reflected different leader sequences, antigen forms, and stabilization vectors (Fig. 1b). 1Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. 2Janssen Vaccines and Prevention BV, Leiden, Netherlands. 3Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA. 4Massachusetts Institute of Technology, Cambridge, MA, USA. 5University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 6Harvard Medical School, Boston, MA, USA. 7Bioqual, Rockville, MD, USA. 8Children’s Hospital, Boston, MA, USA. 9Massachusetts Consortium on Pathogen Readiness, Boston, MA, USA. 10These authors contributed equally: Noe B. Mercado, Roland Zahn, Frank Wegmann, Carolin Loos, Abishek Chandrashekar, Jingyou Yu, Jinyan Liu, Lauren Peter, Katherine McMahan, Lisa H. Tostanoski, Xuan He, David R. Martinez. 11These authors jointly supervised this work: Hanneke Schuitemaker, Dan H. Barouch. ✉e-mail: [email protected] Nature | www.nature.com | 1 Article We immunized 52 adult rhesus macaques, 6-12 years old, with controls, although protection was optimal with Ad26-S.PP (Fig. 4d). Ad26 vectors expressing tPA.S (N=4), tPA.S.PP (N=4), S (N=4), S.dCT All vaccinated animals showed no detectable infectious virus in NS by (N=4), tPA.WT.S (N=4), S.dTM.PP (N=6), S.PP (N=6), and sham controls PFU assays (Extended Data Fig. 5). (N=20). Animals received a single immunization of 1011 viral particles A comparison of peak viral loads in the vaccinated animals suggested (vp) Ad26 vectors by the intramuscular route without adjuvant at that protection in BAL was generally more robust than in NS (Fig. 5). The week 0. We observed RBD-specific binding antibodies by ELISA in 31 Ad26-S.PP vaccine provided complete protection in both the lower and of 32 vaccinated animals by week 2 and in all vaccinated animals by upper respiratory tract with the exception of one animal that showed week 4 (Fig. 2a). Neutralizing antibody (NAb) responses were assessed a low amount of virus in NS, and resulted in >3.2 and >3.9 log10 reducW- W using both a pseudovirus neutralization assay9,10,16 (Fig. 2b) and a live tions of median peak sgRNA in BAL and NS, respectively, as compared 9,10,17,18 virus neutralization assay (Fig. 2c). NAb titers as measured by with sham controls (P<0.0001 and P<0.0001, respectively,E two-sided E both assays were observed in the majority of vaccinated animals at Mann-Whitney tests) (Fig. 5). Among the 32 vaccinated macaques, 17 week 2 and generally increased by week 4. The Ad26-S.PP vaccine animals were completely protected and had no detectableI sgRNA in I elicited the highest pseudovirus NAb titers (median 408; range 208- BAL or NS following challenge, and 5 additional animalsV had no sgRNA V 643) and live virus NAb titers (median 113; range 53-233) at week 4 in BAL but showed some virus in NS. (P<0.05, two-sided Mann-Whitney tests). Pseudovirus NAb titers cor- related with both ELISA titers and live virus NAb titers (P<0.0001, E E R=0.8314 and P<0.0001, R=0.8427, respectively, two-sided Spearman Immune Correlates of Protection rank-correlation tests; Extended Data Fig. 1). Median NAb titers in the The size of this study and the variabilityR in outcomes with the differ- R Ad26-S.PP vaccinated macaques were 4-fold higher than median NAb ent vaccine constructs facilitated an immune correlates analysis.