Measurement of Adenovirus-Based Vector Heterogeneity.

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.

Development of Monoclonal Antibodies to Detect for SARS-CoV-2 Proteins.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection.

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis.

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

Application of spectral library prediction for parallel reaction monitoring of viral peptides.

A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future.

Wearable Collector for Noninvasive Sampling of SARS-CoV-2 from Exhaled Breath for Rapid Detection.

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.

Molecular Diagnostic Tools for the Detection of SARS-CoV-2.

The pandemic causing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally infected more than 50 million people and ∼1.2 million have succumbed to this deadly pathogen. With the vaccine trials still in clinical phases, mitigation of Coronavirus Disease 2019 (COVID-19) relies primarily on robust virus detection methods and subsequent quarantine measures. Hence, the importance of rapid, affordable and reproducible virus testing will serve the need to identify and treat infected subjects in a timely manner. Based on the type of diagnostic assay, the primary targets are viral genome (RNA) and encoded proteins. Currently, COVID-19 detection is performed using various molecular platforms as well as serodiagnostics that exhibit approximately 71% sensitivity. These methods encounter several limitations including sensitivity, specificity, availability of skilled expertise and instrument access. Saliva-based COVID-19 diagnostics are emerging as a superior alternative to nasal swabs because of the ease of sample collection, no interaction during sampling, and high viral titers during early stages of infection. In addition, SARS-CoV-2 is detected in the environment as aerosols associated with suspended particulate matter. Designing virus detection strategies in diverse samples will allow timely monitoring of virus spread in humans and its persistence in the environment. With the passage of time, advanced technologies are overcoming limitations associated with detection. Enhanced sensitivity and specificity of next-generation diagnostics are key features enabling improved prognostic care. In this comprehensive review, we analyze currently adopted advanced technologies and their concurrent use in the development of diagnostics for SARS-CoV-2 detection.

Aptamers: The Powerful Molecular Tools for Virus Detection.

Aptamers are short single-stranded DNA or RNA oligonucleotides selected by the technique of systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have been demonstrated to bind various targets from small-molecule to cells or even tissues in the way of antibodies. Thus, they are called chemical antibodies. We summarize and evaluate recent developments in aptamer-based sensors (for short aptasensors) for virus detection in this review. These aptasensors are mainly classified into optical and electronic aptasensors based on the type of transducer. Nowadays, the smartphone has become the most widely used mobile device with billions of users worldwide. Considering the ongoing COVID-19 outbreak, smartphone-based aptasensors for a portable and point-of-care test (POCT) of COVID-19 detection will be of great importance in the future.

Graphene: A Disruptive Opportunity for COVID-19 and Future Pandemics?

The graphene revolution, which has taken place during the last 15 years, has represented a paradigm shift for science. The extraordinary properties possessed by this unique material have paved the road to a number of applications in materials science, optoelectronics, energy, and sensing. Graphene-related materials (GRMs) are now produced in large scale and have found niche applications also in the biomedical technologies, defining new standards for drug delivery and biosensing. Such advances position GRMs as novel tools to fight against the current COVID-19 and future pandemics. In this regard, GRMs can play a major role in sensing, as an active component in antiviral surfaces or in virucidal formulations. Herein, the most promising strategies reported in the literature on the use of GRM-based materials against the COVID-19 pandemic and other types of viruses are showcased, with a strong focus on the impact of functionalization, deposition techniques, and integration into devices and surface coatings.

Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients.

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.

Proteomics in the COVID-19 Battlefield: First Semester Check-Up.

Proteomics offers a wide collection of methodologies to study biological systems at the finest granularity. Faced with COVID-19, the most worrying pandemic in a century, proteomics researchers have made significant progress in understanding how the causative virus hijacks the host's cellular machinery and multiplies exponentially, how the disease can be diagnosed, and how it develops, as well as its severity predicted. Numerous cellular targets of potential interest for the development of new antiviral drugs have been documented. Here, the most striking results obtained in the proteomics field over this first semester of the pandemic are presented. The molecular machinery of SARS-CoV-2 is much more complex than initially believed, as many post-translational modifications can occur, leading to a myriad of proteoforms and a broad heterogeneity of viral particles. The interplay of protein-protein interactions, protein abundances, and post-translational modifications has yet to be fully documented to provide a full picture of this intriguing but lethal biological threat. Proteomics has the potential to provide rapid detection of the SARS-CoV-2 virus by mass spectrometry proteotyping, and to further increase the knowledge of severe respiratory syndrome COVID-19 and its long-term health consequences.

Diagnosing COVID-19: The Disease and Tools for Detection.

COVID-19 has spread globally since its discovery in Hubei province, China in December 2019. A combination of computed tomography imaging, whole genome sequencing, and electron microscopy were initially used to screen and identify SARS-CoV-2, the viral etiology of COVID-19. The aim of this review article is to inform the audience of diagnostic and surveillance technologies for SARS-CoV-2 and their performance characteristics. We describe point-of-care diagnostics that are on the horizon and encourage academics to advance their technologies beyond conception. Developing plug-and-play diagnostics to manage the SARS-CoV-2 outbreak would be useful in preventing future epidemics.

Urgent need of rapid tests for SARS CoV-2 antigen detection: Evaluation of the SD-Biosensor antigen test for SARS-CoV-2.

At the time of writing, FIND has listed four CE-marked SARSCoV-2 antigen tests. We evaluated the recently CE-approved rapid POCT SD-Biosensor for SARS-CoV-2 nucleoprotein detection in nasopharyngeal secretions from 330 patients admitted to the Emergency Room for a suspect of COVID-19 and travelers returning home from high risk countries. Sensitivity, specificity, accuracy, negative and predictive values were consistent with the use of the test to mass-screening for SARS-CoV-2 surveillance.

SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene.

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.

Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay.

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.

Cytologic and molecular correlates of SARS-CoV-2 infection of the nasopharynx.

Infection by SARS-CoV-2 commonly begins in the nasopharynx, and the cytologic and molecular correlates are not characterized. Fifty-eight cytologic preps (20 oral and 38 from the nasopharynx) were obtained from ten patients and analyzed in a blinded fashion for SARS-CoV-2 spike and envelope protein by immunohistochemistry and viral RNA by in situ hybridization. qRTPCR identified three positive cases and seven controls; the three cases reported mild symptoms that resolved in 2-3 days. Blinded analyses confirmed the presence of the SARS-CoV-2 spike and envelope proteins and viral RNA in the three cases and viral absence in the seven controls. A signal for the positive cases was evident in each nasopharyngeal and none of the oral samples. Viral RNA/proteins localized exclusively to glandular cells and was present in high copy number. Blinded analysis of the cytology documented that the glandular cells infected by SARS-CoV-2 showed marked degeneration with ciliocytophthoria; viral inclusions were not evident. Co-expression analysis showed viral infected cells had increased apoptosis, marked by strong expression of activated caspase 3. Weekly serial testing of two of the cases showed persistence of productive viral infection for up to 2 weeks after symptom onset. It is concluded that the target cell of SARS-CoV-2 in the head and neck region is the glandular cell of the nasal passages, that viral infection is lytic and associated with high copy number that facilitates viral spread. The method outlines a simple, rapid test for productive SARS-CoV-2 based on immunohistochemistry or in situ hybridization of the glandular cells from the nasopharynx.

Four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis.

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established.

Molecular Diagnosis of COVID-19: Challenges and Research Needs.

Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.