Lyophilized, thermostable Spike or RBD immunogenic liposomes induce protective immunity against SARS-CoV-2 in mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide viral pandemic leading to global efforts to produce and distribute effective vaccines that prevent coronavirus virus disease 2019 (COVID-19) (1). The Spike protein and, more specifically, its receptor-binding domain (RBD) on the virus surface are responsible for binding to human angiotensin-converting enzyme 2 (hACE2) on the host cell. Hence, because of their central role in viral entry, the Spike and the RBD are established immunogens in SARS-CoV-2 vaccines (2, 3). mRNA-based and viral-vectored vaccines encoding the Spike protein have gained regulatory approvals and are being massively deployed presently. Despite their excellent protective efficacy against SARS-CoV-2, mRNA vaccines have well-known limited thermostability, and thus, global distribution is complicated by the requirement of ultracold freezing storage temperatures (4). As COVID-19 vaccines are needed worldwide to immunize sufficient populations to induce global herd immunity, including in low- and middle-income countries, there is more demand for vaccine doses than at any other time in history (5). A potent vaccine that is effective with a lower amount of antigen per dose would increase manufacturing and distribution capacity, facilitating the supply of global needs during the pandemic.This research was supported by the NIH (R43AI165089), the European Union’s Horizon 2020 research and innovation program under a Marie Skłodowska-Curie grant (E.R.), the CIFAR Azrieli Global Scholar program (J.P.-J.), the Ontario Early Researcher Awards program (J.P.-J.), and the Canada Research Chairs program (J.P.-J.). Cryo-EM images were collected at the Facility for Electron Microscopy Research (FEMR) at McGill University. FEMR is supported by the Canadian Foundation for Innovation, the Quebec government, and McGill University.Peer reviewe

Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases

Role of Era in assembly and homeostasis of the ribosomal small subunit

Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.National Institutes of Health (U.S.) (Grant 5R00AG050749

Antibody induction in mice by liposome-displayed recombinant enterotoxigenic Escherichia coli (ETEC) colonization antigens

Background: Enterotoxigenic Escherichia coli (ETEC) strains cause infectious diarrhea and colonize host intestine epithelia via surface-expressed colonization factors. Colonization factor antigen I (CFA/I), a prevalent ETEC colonization factor, is a vaccine target since antibodies directed to this fimbria can block ETEC adherence and prevent diarrhea. Methods: Two recombinant antigens derived from CFA/I were investigated with a vaccine adjuvant system that displays soluble antigens on the surface of immunogenic liposomes. The first antigen, CfaEB, is a chimeric fusion protein comprising the minor (CfaE) and major (CfaB) subunits of CFA/I. The second, CfaEad, is the adhesin domain of CfaE. Results: Owing to their His-tag, recombinant CfaEB and CfaEad, spontaneously bound upon admixture with nanoliposomes containing cobalt-porphyrin phospholipid (CoPoP), as well as a synthetic monophosphoryl lipid A (PHAD) adjuvant. Intramuscular immunization of mice with sub-microgram doses CfaEB or CfaEad admixed with CoPoP/PHAD liposomes elicited serum IgG and intestinal IgA antibodies. The smaller CfaEad antigen benefitted more from liposome display. Serum and intestine antibodies from mice immunized with liposome-displayed CfaEB or CfaEad recognized native CFA/I fimbria as evidenced by immunofluorescence and hemagglutination inhibition assays using the CFA/I-expressing H10407 ETEC strain. Conclusion: These data show that colonization factor-derived recombinant ETEC antigens exhibit immunogenicity when delivered in immunogenic particle-based formulations

Acute pancreatitis in liver transplant hospitalizations: Identifying national trends, clinical outcomes and healthcare burden in the United States

Background: Acute pancreatitis (AP) in liver transplant (LT) recipients may lead to poor clinical outcomes and development of severe complications. Aim: We aimed to assess national trends, clinical outcomes, and the healthcare burden of LT hospitalizations with AP in the United States (US). Methods: The National Inpatient Sample was utilized to identify all adult (≥ 18 years old) LT hospitalizations with AP in the US from 2007-2019. Non-LT AP hospitalizations served as controls for comparative analysis. National trends of hospitalization characteristics, clinical outcomes, complications, and healthcare burden for LT hospitalizations with AP were highlighted. Hospitalization characteristics, clinical outcomes, complications, and healthcare burden were also compared between the LT and non-LT cohorts. Furthermore, predictors of inpatient mortality for LT hospitalizations with AP were identified. All P values ≤ 0.05 were considered statistically significant. Results: The total number of LT hospitalizations with AP increased from 305 in 2007 to 610 in 2019. There was a rising trend of Hispanic (16.5% in 2007 to 21.1% in 2018, P-trend = 0.0009) and Asian (4.3% in 2007 to 7.4% in 2019, p-trend = 0.0002) LT hospitalizations with AP, while a decline was noted for Blacks (11% in 2007 to 8.3% in 2019, P-trend = 0.0004). Furthermore, LT hospitalizations with AP had an increasing comorbidity burden as the Charlson Comorbidity Index (CCI) score ≥ 3 increased from 41.64% in 2007 to 62.30% in 2019 (P-trend \u3c 0.0001). We did not find statistically significant trends in inpatient mortality, mean length of stay (LOS), and mean total healthcare charge (THC) for LT hospitalizations with AP despite rising trends of complications such as sepsis, acute kidney failure (AKF), acute respiratory failure (ARF), abdominal abscesses, portal vein thrombosis (PVT), and venous thromboembolism (VTE). Between 2007-2019, 6863 LT hospitalizations with AP were compared to 5649980 non-LT AP hospitalizations. LT hospitalizations with AP were slightly older (53.5 vs 52.6 years, P = 0.017) and had a higher proportion of patients with CCI ≥ 3 (51.5% vs 19.8%, P \u3c 0.0001) compared to the non-LT cohort. Additionally, LT hospitalizations with AP had a higher proportion of Whites (67.9% vs 64.6%, P \u3c 0.0001) and Asians (4% vs 2.3%, P \u3c 0.0001), while the non-LT cohort had a higher proportion of Blacks and Hispanics. Interestingly, LT hospitalizations with AP had lower inpatient mortality (1.37% vs 2.16%, P = 0.0479) compared to the non-LT cohort despite having a higher mean age, CCI scores, and complications such as AKF, PVT, VTE, and the need for blood transfusion. However, LT hospitalizations with AP had a higher mean THC ($59596 vs$50466, P = 0.0429) than the non-LT cohort. Conclusion: In the US, LT hospitalizations with AP were on the rise, particularly for Hispanics and Asians. However, LT hospitalizations with AP had lower inpatient mortality compared to non-LT AP hospitalizations