ABSTRACT
A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).
Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Disease Models, Animal , SARS-CoV-2/immunology , Aging/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/chemistry , Antigens, Viral/genetics , Antigens, Viral/immunology , BNT162 Vaccine , COVID-19/blood , COVID-19/therapy , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , Cell Line , Clinical Trials as Topic , Female , Humans , Immunization, Passive , Internationality , Macaca mulatta/immunology , Macaca mulatta/virology , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Multimerization , RNA, Viral/analysis , Respiratory System/immunology , Respiratory System/virology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Solubility , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , Vaccination , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , COVID-19 Serotherapy , mRNA VaccinesABSTRACT
The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 Å resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins.
Subject(s)
Archaeal Proteins/chemistry , CRISPR-Associated Proteins/chemistry , Euryarchaeota/metabolism , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Archaeal Proteins/metabolism , CRISPR-Associated Proteins/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Point Mutation , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Archaeal/genetics , RNA, Archaeal/metabolism , RNA-Binding Proteins/metabolismABSTRACT
The methanogenic archaeon Methanopyrus kandleri grows near the upper temperature limit for life. Genome analyses revealed strategies to adapt to these harsh conditions and elucidated a unique transfer RNA (tRNA) C-to-U editing mechanism at base 8 for 30 different tRNA species. Here, RNA-Seq deep sequencing methodology was combined with computational analyses to characterize the small RNome of this hyperthermophilic organism and to obtain insights into the RNA metabolism at extreme temperatures. A large number of 132 small RNAs were identified that guide RNA modifications, which are expected to stabilize structured RNA molecules. The C/D box guide RNAs were shown to exist as circular RNA molecules. In addition, clustered regularly interspaced short palindromic repeats RNA processing and potential regulatory RNAs were identified. Finally, the identification of tRNA precursors before and after the unique C8-to-U8 editing activity enabled the determination of the order of tRNA processing events with termini truncation preceding intron removal. This order of tRNA maturation follows the compartmentalized tRNA processing order found in Eukaryotes and suggests its conservation during evolution.
