The coronavirus disease 2019 (COVID-19) pandemic caused by the emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health, and there is an urgent need to develop safe and effective vaccines. Here, we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV-2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies that neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, T helper (Th)1-dominated cellular response. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV-2-neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at a massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of the GRAd-COV2 vaccine in a currently ongoing phase I clinical trial (ClinicalTrials.gov: NCT04528641).

The COVID-19 pandemic caused by the emergent SARS-CoV-2 coronavirus threatens global public health and there is an urgent need to develop safe and effective vaccines. Here we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies which neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, Th1-dominated cellular response in the periphery and in the lung. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV2 neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of GRAd-COV2 vaccine in a currently ongoing Phase I clinical trial (NCT04528641).

Lipopolysaccharide (LpS) is a critical component of the outer membrane (oM) of many Gramnegative bacteria. LpS is translocated to the oM by the LpS transport (Lpt) system. in the human pathogen Pseudomonas aeruginosa, the periplasmic Lpt component, LptH, is essential for LpS transport, planktonic and biofilm growth, OM stability and infectivity. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization. The relevance of these sites for LptH activity was verified through plasmidmediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. complementation and protein expression analyses provided evidence that all mutated sites are important for LptH activity in vivo. It was observed that the lptH conditional mutant overcomes the lethality of nonfunctional lptH variants through RecA-mediated homologous recombination between the wild-type lptH gene in the genome and mutated copies in the plasmid. finally, biochemical assays on purified recombinant proteins showed that some LptH variants are indeed specifically impaired in oligomerization, while others appear to have defects in protein folding and/or stability. The cell envelope of diderm (Gram-negative) bacteria consists of two concentric membranes, the inner (IM) and outer membrane (OM), which confine an aqueous compartment, the periplasmic space, in which a thin layer of peptidoglycan is embedded. While the IM is a typical phospholipids bilayer, the OM of most diderm bacteria is an asymmetric membrane composed of lipopolysaccharide (LPS) and phospholipids in the outer and inner leaflets, respectively 1. LPS is a negatively charged glycolipid that forms a tightly packed layer at the cell surface. The LPS layer is important for the structural stability of the OM, and provides an effective permeability barrier to the entry of potentially noxious compounds 2. LPS is synthesized in the cytoplasm, matured in the periplasm and translocated to the OM by the Lpt (Lipopolysaccharide transport) system that, in the model organism Escherichia coli, is composed of seven essential proteins (LptABCDEFG). The Lpt protein complex spans the entire cell envelope and consists of two subassemblies, LptB 2 CFG at the IM and LptDE at the OM, connected by the periplasmic protein LptA 3-5. LptB 2 FG is an ATP-binding cassette (ABC) transporter that, in association with the bitopic protein LptC, powers LPS transport to the cell surface, while the β-barrel protein LptD and the lipoprotein LptE constitute the OM translocon that inserts LPS into the outer leaflet of the OM 3-5 (Fig. 1A).