Summary SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4 + and CD8 + T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro . Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Malignant glioma constitutes one of the fatal primary brain tumors in adults. Such poor prognosis calls for a better understanding of cancer-related signaling pathways of this disease.Here we elucidate a MYC-miRNA-MXI1 feedback loop that regulates proliferation and tumorigenesis in glioma. MYC suppressed MXI1 expression via microRNA-155 (miR-155) and the microRNA-23a$27a$24-2 cluster (miR-23a cluster), whereas MXI1, in turn, inhibited MYC expression by binding to its promoter. Overexpression of miR-155 and the miR-23a cluster promoted tumorigenesis in U87 glioma cells. Furthermore, fat mass and obesity-associated protein (FTO), an N 6 -methyladenosine (m 6 A) RNA demethylase, regulated the loop by targeting MYC. The ethyl ester form of meclofenamic acid (MA2) inhibited FTO and enhanced the effect of the chemotherapy drug temozolomide on suppressing proliferation of glioma cells and negatively regulated the loop. These data collectively highlight a key regulatory circuit in glioma and provide potential targets for clinical treatment.Significance: These findings elucidate a novel feedback loop that regulates proliferation in glioma and can be targeted via inhibition of FTO to enhance the efficacy of temozolomide.

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is a key target for vaccine development efforts. Stabilized S ectodomain constructs have been developed that mimic the native spike, bind the ACE-2 receptor 1,2 and present epitopes for neutralizing antibodies on their surface [3][4][5][6] . The so-called '2P' S ectodomain construct (2P S) comprises residues 1-1,208 of SARS-CoV-2 S and contains two proline (2P) substitutions in the C-terminal S2 domain designed to stabilize the prefusion S conformation; a C-terminal foldon trimerization motif and a mutation that abrogates the furin-cleavage site 1 (Fig. 1a). This and similar constructs have been widely used for structural biology and vaccine studies [1][2][3]7,8 . Purified S ectodomain proteins 9 are assessed for quality control by SDS-PAGE, size exclusion chromatography (SEC), differential scanning fluorimetry (DSF) 10 and negative-stain electron microscopy (NSEM). The last technique has been particularly informative because it reveals the structural integrity of individual molecules, allowing us to examine preparations that look similar by using bulk methods such as SDS-PAGE and SEC (Supplementary Fig. 1). The observed variability between preparations indicates a fragile S ectodomain, and measures to overcome the issue have been previously reported 11,12 .Here we link the apparent fragility of 2P S to its rapid denaturation on storage at 4 °C (Fig. 1b). We followed the structural, biophysical and antigenic properties of 2P S stored under different temperature conditions (Fig. 1, Extended Data Figs. 1 and 2, Supplementary Tables 1 and 2 and Supplementary Figs. 2-6). 2P S was produced in 293F cells at 37 °C and purified at room temperature within 6-8 h (Supplementary Fig. 1). We performed NSEM analysis of 2P S incubated at different temperatures (Fig. 1b and Extended Data Fig. 1d,e). Freshly prepared 2P S samples assessed on the same day they were purified showed on average 75% well-formed spikes, with characteristic kite-shaped morphology on NSEM micrographs

SummaryThe impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine development. Here we report an unexpected cold sensitivity of a stabilized SARS-CoV-2 ectodomain construct currently being widely used for immunogen design. We found that when stored at 22 or 37 °C for 1 week, the S-protein displayed well-ordered trimeric spikes by negative stain electron microscopy. However, storage at 4 °C reduced the trimeric spikes to <10%, accompanied by decreased stability and enhanced exposure of the ACE-2 receptor binding site. Well-formed S particles could be recovered from cold-stored samples by a brief incubation at 37 °C. Our results will have broad impact on structural, functional and vaccine studies using the SARS-CoV-2 S ectodomain.HighlightsSARS-CoV-2 S ectodomain construct, widely used for vaccine studies, exhibits cold sensitivity.Negative stain electron microscopy shows disintegration of spike structure upon storage at 4 °C.Differential scanning calorimetry measurements confirm destabilization by cold.Cold storage alters antigenicity of SARS-CoV-2 spike.Brief incubation at 37 °C restored spike integrity after cold-storage.

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single−molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env. Structural analysis by single−particle cryoelectron microscopy performed on the BG505 SOSIP mutant Env proteins shows rearrangements in the gp120 topological layer contacts with gp41. Displacement of a conserved tryptophan (W571) from its typical pocket in these Env mutants renders the Env insensitive to CD4 binding. These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.