J.D. collected from December 2019 to June 2022 used in this study were obtained from GISAID EpiCoV database (https://www.epicov.org/epi3/frontend#). Representative spike sequences from sarbecoviruses used in this study were obtained from NCBI Virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/).?Source data are provided OTSSP167 with this paper. Abstract As SARS-CoV-2 Omicron and other variants of concern (VOCs) continue spreading worldwide, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with potency against diverse VOCs including Omicron subvariants BA.1, BA.2 and BA.4/5, SARS-CoV-1, and major sarbecoviruses. Crystal structure analysis of one representative nanobody, 3-2A2-4, discovers a highly conserved epitope located between the cryptic and the outer face of the receptor binding domain (RBD), distinctive from the receptor ACE2 binding site. Cryo-EM and biochemical evaluation reveal that 3-2A2-4 interferes structural alteration of RBD required for ACE2 binding. Passive delivery of 3-2A2-4 protects K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. Identification of these unique nanobodies will inform the development of next generation antibody therapies and OTSSP167 design of pan-sarbecovirus vaccines. Subject terms: Immunology, Structural biology, SARS-CoV-2 The authors identify nanobodies from immunized alpaca with broadly neutralizing activity against SARS-CoV-1, SARS-CoV-2 variants, and major sarbecoviruses. One representative nanobody binds to a highly conserved epitope on RBD and protects K18-hACE2 mice from Omicron and Delta infection. Introduction As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to rage globally, we have been witnessing the rapid emergence OTSSP167 and turnover of multiple variants of concerns (VOCs) such as Alpha (B.1.1.7) initially found in the United Kingdom; Beta (B.1.351) in South Africa; Gamma (P.1) in Brazil; Delta (B.1.617.2) in India; and Omicron subvariants in Botswana and South Africa (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/). These VOCs are not only associated with steeply increased new infections among unvaccinated but also break-through infections among the infected and vaccinated individuals1C4. Increasing evidence suggests that substantial changes in their antigenic properties have facilitated these VOCs to escape from serum neutralization of convalescent and vaccinated individuals5C9. As a result, efficacies of all vaccine modalities as well as many therapeutic antibodies approved for emergency use authorization (EUA) have been severely compromised, particularly toward Omicron subvariants BA.1, BA.2, BA.3, and BA.4/5, followed by Beta, Delta, Gamma, and to the least extent by Alpha7,10C12. Omicron subvariants are perhaps the most insidious as they generally cause milder symptoms but carries the exceptionally high viral load in the upper respiratory tract with extremely high efficiency in transmission13C15. As quiet as it seems, Omicron subvariant BA.1, then BA.2, and now BA.4/5 have been actively replacing other VOCs and local variants to become the most dominant variant in many parts of the OTSSP167 world. Development of broader and more effective therapies and vaccines against these Omicron subvariants has therefore become an urgent and global priority. One striking aspect of Omicron is the largest number of mutations found in the spike (S) protein among the VOCs identified thus far (https://www.gisaid.org), although the origins and mechanism of their accumulations remain unclear16,17. At least 35 substitutions were found in the S protein of Omicron compared to the prototype strain from Wuhan, China. Of which, about 15 are located in the RBD and 8 in the N-terminal domain (NTD), although the exact OTSSP167 number of substitutions vary among different subvariants (https://www.gisaid.org). BA.1 and BA.2 are two early subvariants of Omicron that emerged around the end of 2021 and have since then been rapidly spreading worldwide. However, BA.4 and BA.5 subvariants, found in early April 2022 in Gauteng of South Africa, are actively replacing BA.1 and BA.2 and fueling the current wave of new and breakthrough infections in many parts of the world18. Genetically, BA.1 is rather unique while BA.2 (and its 12th and 75th lineage BA.2.12.1 and BA.2.75, respectively), BA.4, and BA.5 are highly related19. BA.3 appeared to be the mosaic between BA.1 and BA.2 (https://www.gisaid.org). BA.4 and BA.5 are believed to EIF4G1 have evolved from BA.2 and share identical S sequences, thus frequently referred to as BA.4/5. Recently, several elegant studies have pinpointed a few key substitutions in the S protein that are responsible for neutralization escape, and many of which.