Antigen-specific CD8+ T cells were detected using H2-Db S538C546-specific dextramers (Immudex). full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model. The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants. Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice. Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for Ciclopirox further preclinical studies of d16 as a prototypic vaccine strain, to which new features might be introduced to improve safety, transmissibility, immunogenicity and efficacy. value of 0.05 . The complex heatmap R package  was used to visualize the top 10 differentially expressed genes normalized by score for the d16 vs. WT?+?mock, WT vs. d16?+?mock, and mock vs. D16?+?WT comparisons. Effects on vital biological gene functions were identified by performing enrichment analysis using the clusterProfiler R package  with the default parameters of a false discovery rate RPA3 0.05 and 1000 permutations.?The RNA-seq data were deposited in Mendeley Data under the DOI of 10.17632/d/dnscfxkddd.1.? Infection of Syrian hamsters and K18-hACE2 transgenic mice All experimental protocols were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong and were performed according to the standard operating procedures of the biosafety level 3 animal facilities (reference code: CULATR 5754-21) in adherence with the NIH Guide for the Care and Use of Laboratory Animals. Animal infection experiments were performed as previously described with slight modifications . Syrian hamsters (6C8 weeks old) were obtained from the Chinese University of Hong Kong Laboratory Animal Service through the Center for Comparative Medicine Research (CCMR) of the University of Hong Kong. K18-hACE2 transgenic mice were bred, raised in the CCMR and delivered upon reaching 6C8 weeks of age. The animals were raised in a biosafety level 2 facility with ad libitum access to standard pellet feed Ciclopirox and water. During vaccination, hamsters were first anesthetized with intraperitoneal ketamine (200?mg per kg) and xylazine (10?mg per kg) administration and then intranasally inoculated with WT or mutant SARS-CoV-2 at 105 PFU. PBS served as a diluent to obtain the desired concentrations from viral stocks. A challenge dose of 105 PFU SARS-CoV-2 (clinical isolate HK-13) was used to challenge vaccinated or sham-vaccinated hamsters . For K18-hACE2 transgenic mice, a dose of 103 PFU recombinant virus (d16) was used for vaccination, whereas a dose of 104 PFU SARS-CoV-2 (clinical isolate HK-13) was inoculated for antiviral evaluation. Body weights were continuously monitored for 14 days after infection or until animal death. For virological and histopathological examinations, animals were sacrificed at 4 dpi, and their organs, tissues, Ciclopirox nasal wash and blood were collected for analyses. The two halves of harvested tissues were used for viral titration by RTCqPCR or a plaque assay and for histopathological analysis following fixation in 10% PBS-buffered formaldehyde, as we previously described . For histopathological assessment, a semiquantitative system was employed to evaluate the relative degree of inflammation and tissue damage . Inflammation was scored as follows: 0, no inflammation; 1, perivascular cuff of inflammatory cells; 2, mild inflammation (extending through 25% of the lung); 3, moderate inflammation (25C50% of the lung); and 4, severe inflammation involving over half of the lung. The examination of tissue slides was carried out in a single-blind manner. Ciclopirox Antibody tests An enzyme-based immunoassay was performed as previously described [59, 63]. Ninety-six-well plates (Nunc, Rochester, NY, USA) were coated with 0.05?g/well SARS-CoV-2 RBD in 100?l of 0.05?M NaHCO3 (pH 9.6) overnight at 4?C. After blocking, 100?l of heat-inactivated serum samples was serially diluted before being added to the wells and incubated at 37?C for 1?h. RBD-binding antibodies were detected using a horseradish peroxidase-conjugated goat anti-hamster IgG antibody (A18895 from Invitrogen, Waltham, MA, USA), anti-mouse IgG antibody (Life Technology 626720) or anti-mouse IgA antibody (SigmaCAldrich SAB5600003), followed by the addition of a diluted 3,3,5,5-tetramethylbenzidine (TMB) single solution (Invitrogen) and 1?M HCl. The optical density at 450?nm (OD450) was measured using a microplate reader. The sVNT was performed using cPass (GenScript) according to the.