Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.

The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.

A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs.

Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies.

Promising Tools to Facilitate the Implementation of TDM of Biologics in Clinical Practice.

Therapeutic drug monitoring (TDM) of biologics-encompassing the measurement of (trough) concentrations and anti-drug antibodies-is emerging as a valuable tool for clinical decision making. While this strategy needs further validation, attention on its implementation into the clinic is warranted. Rapid testing and easy sampling are key to its implementation. Here, we aimed to evaluate the feasibility and volunteers' perception of home microsampling for quantification of adalimumab (ADM) concentrations in psoriasis patients. In addition, we compared lateral flow testing (LFT) with enzyme-linked immunosorbent assay (ELISA). Patients participating in the SUPRA-A study (clinicaltrials.gov NCT04028713) were asked to participate in a substudy where volumetric absorptive microsampling (VAMS) was performed at home. At three time points, whole blood and corresponding serum samples were collected for ADM measurement using an in-house ELISA. In addition, the patients' perspective on microsampling was evaluated via a questionnaire. LFT-obtained ADM concentrations agreed very well with ELISA results (Pearson's correlation = 0.95 and R2 = 0.89). ADM concentrations determined in both capillary (via finger prick) and corresponding venous blood VAMS samples correlated strongly with serum concentrations (Pearson's correlation = 0.87). Our preliminary data (n = 7) on rapid testing and home-based microsampling are considered promising with regard to TDM implementation for adalimumab, warranting further research.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.