ABSTRACT
Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity, adverse events that may be partly due to systemic off target expression of the immunogen, and a very limited understanding of the mechanisms responsible for the frozen storage requirement. We applied a new rational design process to identify a novel multiprotic ionizable lipid, called C24, as the key component of the mRNA LNP delivery system. We show that the resulting C24 LNP has a multistage protonation behavior resulting in greater endosomal protonation and greater translation of an mRNA-encoded luciferase reporter after intramuscular (IM) administration compared to the standard reference MC3 LNP. Off-target expression in liver after IM administration was reduced 6 fold for the C24 LNP compared to MC3. Neutralizing titers in immunogenicity studies delivering a nucleoside-modified mRNA encoding for the diproline stabilized spike protein immunogen were 10 fold higher for the C24 LNP versus MC3, and protection against viral challenge in a SARS-CoV-2 mouse model occurred at a very low 0.25 µg prime/boost dose of the same immunogen in the C24 LNP. Injection site inflammation was notably reduced for C24 compared to MC3. In addition, we found the C24 LNP to be entirely stable in bioactivity and mRNA integrity when stored at 4 ºC for at least 19 days. Storage at higher temperatures reduced both bioactivity and mRNA integrity, but less so for C24 than MC3, and in a manner consistent with the phosphodiester transesterification reaction mechanism of mRNA cleavage. The higher potency, lower injection site inflammation, and higher stability of the C24 LNP present important advancements in the evolution mRNA vaccine delivery.
ABSTRACT
Introduction: Coronavirus disease 2019 (COVID-19) is a systemic disease characterized by a disproportionate inflammatory response in the acute phase. However, long-term clinical, functional, and metabolic consequences are still unknown. This study sought to identify clinical sequelae and its potential intrinsic mechanism among COVID-19 survivors in the follow-up. Methods: We conducted a prospective single-center study (NCT04689490) of previously hospitalized COVID-19 patients with and without dyspnea during mid-term follow-up, an outpatient asymptomatic control group was also evaluated. They underwent serial testing with cardio-pulmonary exercise test (CPET), transthoracic echocardiogram, pulmonary lung test, six-minute walking test, serum biomarker analysis and quality of life questionaries.Results: Patients with dyspnea (n=41, 58.6%), compared with asymptomatic (n=29, 41.4%), had a higher proportion of females (73.2% vs. 51.7%; p= 0.065), with comparable age and prevalence of cardiovascular risk factors. There were no significant differences in transthoracic echocardiogram and pulmonary function test, in either group. Patients who referred dyspnea had a significant decline in predicted peak O2 consumption (77.8 [64-92.5] vs. 99 [88-105]: p<0.00; p<0.001), total distance in the 6-minute walking test (535 [467-600] vs. 611 [550-650] meters; p= 0.001), and quality of life (KCCQ-23 60.1±18.6 vs. 82.8±11.3; p<0.001). Additionally, abnormalities in CPET were suggestive of a ventilation/perfusion characterized by impaired ventilatory efficiency (VE/VCO2 slope 32 [28.1-37.4] vs. 29.4 [26.9-31.4]; p= 0.022) and low O2 pulse (9.2 [7.3-11.3] vs. 10.6 [8.7-13.2]; p= 0.013). Interpretation: In this study >50% of COVID-19 survivors present a symptomatic functional impairment irrespective of age or prior hospitalization. Our findings suggest potential ventilation/perfusion mismatch.Funding Statement: The present study was partially granted by Gerencia Regional de Salud de Castilla y León under grant number GRS COVID 111/A/20 and Grant from the Spanish Society of Cardiology: SEC/FEC-INVCLI 20/030Declaration of Interests: None.Ethics Approval Statement: The institutional local ethics committees approved the study protocol (CASVE PI-20-1894) and all patients provided written informed consent before inclusion.
Subject(s)
COVID-19 , Dyspnea , Mastocytosis, SystemicABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the sole causative agent of coronavirus infectious disease-19 (COVID-19). Methods: We performed a retrospective single-center study of consecutively admitted patients between March 1st and May 15th, 2020, with a definitive diagnosis of SARS-CoV-2 infection. The primary endpoint was to evaluate the association of lipid markers with 30-days all-cause mortality in COVID-19. Results: A total of 654 patients were enrolled, with an estimated 30-day mortality of 22.8% (149 patients). Non-survivors had lower total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) levels during the entire course of the disease with complete resolution among survivors. Both showed a significant inverse correlation with inflammatory markers and a positive correlation with lymphocyte count. In a multivariate analysis, LDL-c < 69 mg/dl (hazard ratio [HR] 1.94; 95% confidence interval [CI] 1.14-3.31), C-reactive protein > 88 mg/dl (HR 2.44; 95% CI, 1.41-4.23) and lymphopenia < 1000 cells/ml (HR 2.68; 95% CI, 1.91-3.78) at admission were independently associated with 30-day mortality. This association was maintained 7 days after admission. Conclusion: Hypolipidemia in SARS-CoV-2 infection may be secondary to an immune-inflammatory response, with complete recovery in survivors. Low LDL-c serum levels are independently associated with higher 30-day mortality in COVID-19 patients.