(I) Multifocal minimal to moderate numbers of SARS-CoV-2 N-positive pneumocytes (*) in seven out of eight pooled control animals. those in adult animals. Aged Ad26.COV2.S-vaccinated LTX-401 animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen. Introduction Development of multiple safe and effective vaccines LTX-401 to control the ongoing COVID-19 pandemic (Cucinotta and Vanelli, 2020; World Health Organization, 2020b) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; Wu et al., 2020; Zhu et al., 2020) is a global priority, and several vaccines have already been authorized or approved for use in humans to fight the pandemic (Voysey et al., 2021; Baden et al., 2021; Polack et al., 2020). Ideally, especially in the context of a pandemic, a vaccine provides both early onset of protection and durable protection. The durability of vaccine-elicited protection depends on the capacity of the vaccine platform, specific antigen (design), and vaccination regimen to efficiently stimulate the immune system (Cohen, 2019; Pulendran and Ahmed, 2011) and on several characteristics linked to the recipient of the vaccine (Zimmermann and Curtis, 2019). Age, for instance, plays an important role, as in the elderly the immune response to vaccines is usually reduced in magnitude and duration, potentially resulting in reduced vaccine efficacy (Wagner et al., 2018; Crooke et al., 2019; Gustafson et al., 2020; Weinberger, 2018). Although people of all ages are at risk of contracting COVID-19, the risk of developing severe or critical illness increases markedly with age (Mallapaty, 2020; Centers for Disease Control and Prevention, 2020), warranting the testing of COVID-19 vaccine candidates in different age cohorts. The Ad26.COV2.S vaccine candidate is a replication-incompetent adenovirus 26 (Ad26)Cbased vector encoding the stabilized full-length SARS-CoV-2 spike protein based on LTX-401 the Wuhan-Hu-1 SARS-CoV-2 isolate and containing an aspartic acid (D) residue in amino acidic position 614 (D614; Bos et al., 2020). In preclinical efficacy studies, a single dose of Ad26.COV2.Sprovided robust protection against SARS-CoV-2 challenge (USA-WA1/2020 viral strain, D614) in both upper and lower airways in rhesus macaques (Mercado et al., 2020) and protected Syrian golden hamsters from severe clinical disease (Tostanoski et al., 2020). Protective efficacy against SARS-CoV-2 in nonhuman primates (NHPs) in this and other studies strongly correlated with the presence of virus-binding and neutralizing LTX-401 antibodies in serum (Yu et al., 2020; Mercado et al., 2020; McMahan et al., 2021). These data corroborate previously reported findings on SARS-CoV showing that neutralizing antibody responses against the SARS-CoV spike protein, which binds to the same cellular receptor as SARS-CoV-2 for cell entry (Shan et al., 2020), were associated with protection against SARS-CoV challenge in nonclinical models (Chen et al., 2005). Interim analyses of a Phase 1/2a study showed that Ad26.COV2.S elicits a prompt and robust immune response after a single-dose vaccination in both adults (18C55 yr old) and elderly (65 yr old) humans, as measured up to day 29 after immunization (Sadoff et al., HAS1 2021). Based on these data, the protective efficacy against COVID-19 is currently being evaluated in humans in a Phase 3 LTX-401 one-dose efficacy trial (ENSEMBLE trial, “type”:”clinical-trial”,”attrs”:”text”:”NCT04505722″,”term_id”:”NCT04505722″NCT04505722), for which a median follow-up of participants of 2 mo has shown an early indication of efficacy of 85% against severe/critical COVID-19 in humans (Ledford, 2021). Additionally, a second Phase 3 study (ENSEMBLE 2, “type”:”clinical-trial”,”attrs”:”text”:”NCT04614948″,”term_id”:”NCT04614948″NCT04614948) is currently evaluating vaccine efficacy and durability of a two-dose Ad26.COV2.S regimen as well, as the durability of immunity and efficacy may potentially be enhanced by a second dose. Indeed, in other programs with Ad26-based vaccines, two doses induced higher and more durable immune responses (Geisbert et al., 2011; Callendret et al., 2018; Salisch et al., 2019; Salisch et al., 2021). Here,.