Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

INTRODUCTION: Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. MATERIALS AND METHODS: This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. RESULTS: A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. CONCLUSIONS: Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

Additive Manufacturing for Fabrication of Point-of-Care Therapies in Austere Environments.

INTRODUCTION: Known as the "golden hour," survival of most critically injured patients is highly dependent on providing the required treatment within the first hour of injury. Recent technological advances in additive manufacturing (also known as three-dimensional [3D] printing) allow for austere deployment and point-of-care rapid fabrication of a variety of medical supplies, including human tissues and bioactive bandages, in prolonged field care scenarios. In this pilot project, our aim was to investigate the ability to 3D print a range of potential biomedical supplies and solutions in an austere field environment. MATERIALS AND METHODS: We specifically designed and fabricated novel surgical tools, bioactive bandages, objects (screw and anatomic models), and human meniscal tissue in an austere African desert environment. A total of seven packages were sent using a commercial carrier directly to the end destination. A multi-tool ruggedized 3D printer was used as the manufacturing platform for all objects fabricated downrange. Human mesenchymal stem cells were shipped for 3D bioprinting of human menisci and bioactive bandages. Design and fabrication for all 3D-printed products utilized computer-aided design (CAD) tools. RESULTS: Initial shipment from a single U.S. site to the sub-Saharan Africa location was relatively prompt, taking an average of 4.7 days to deliver three test packages. However, the actual delivery of the seven packages from Orlando, FL, to the same sub-Saharan Africa site took an average of 16 days (range 7-23 days). The ruggedized printer successfully fabricated relevant medical supplies using biocompatible filament, bioink hydrogels, and stem cell-loaded bioinks. This prototype did not, however, have the capacity to provide a sterile environment. A multi-material complete bandage was 3D printed using polyamide polyolefin and cellulose, live cells, neomycin salve, and adhesive. The bandage, wound covering backing, and adhesive backing print took under 2 min to 3D print. Surgical instrument CAD files were based on commercially available medical-grade stainless-steel instruments. The screw CAD file was downloaded from the NIH 3D Print Exchange website. The prints of the two surgical tools and screw using thermoplastic material were successful. Menisci, relatively complex forms of the cartilage, were 3D bioprinted with a gel that held their form well after printing and were then solidified slightly using a cross-linking solution. After 2 min of solidification, it was possible to remove and handle the menisci. CONCLUSION: The current and future challenges of prolonged field care need to be addressed with new techniques, training, and technology. Ruggedized, deployable 3D printers allow for the direct fabrication of medical tools, supplies, and biological solutions for austere use. Delivery of packages can vary, and attention to routes and location is key, especially for transit of time-sensitive perishable supplies such as live cells. The significance of this study provides the real possibility to 3D print "just-in-time" medical solutions tailored to the need of an individual service member in any environment. This is a potentially exciting opportunity to bring critical products to the war front.

Development of a bioreactor for in-vitro compression cycling of tissue engineered meniscal implants.

Injuries to the meniscus are common and can impair physical activities. Bioprinted meniscal tissue offers an attractive alternative to donor tissue for meniscal repair but achieving the strength of native tissue is a challenge. Here we report the development of a tissue engineering bioreactor designed to apply repetitive force which may lead to an increase in the compressive modulus and durability of bioprinted meniscal tissues. The modular bioreactor system is composed of a sterilizable tissue culture vessel together with a dock that applies and measures mechanical force. The culture vessel allows for simultaneous compression cycling of two anatomically sized menisci. Using a hybrid linear actuator with a stepper motor, the dock can apply up to 300 N of force at speeds up to 20 mm/s, corresponding to the upper limits of anatomical force and motion in the knee. An interchangeable 22 N load cell was mated between the culture vessel and the dock to log changes in force. Both the culture vessel and dock are maintained in a standard cell culture incubator to provide heat and CO2, while the dock is powered and controlled externally using a step motor drive and customized software.

Artificial Blood Development Implications for Military Medicine.

Massive hemorrhaging remains the most common cause of preventable battlefield deaths. Blood used for trauma care requires a robust donation network, capacity for long-term storage, and extensive and accurate testing. Bioengineering technologies could offer a remedy to these constraints in the form of blood substitutes-fluids that could be transfused into patients to provide oxygen, carry away waste, and aid in coagulation-that would be used in prolonged casualty care and in far-forward settings, overcoming the obstacles of distance and time. The different molecular properties of red blood cells (RBCs), blood substitutes, and platelet replacements contribute to their respective utilities, and each type is currently represented in ongoing clinical trials. Hemoglobin oxygen carriers (HBOCs) are the most advanced RBC replacements, many of which are currently being evaluated in clinical trials in the United States and other countries. Despite recent advancements, challenges remaining in the development of blood alternatives include stability, oxygen capacity, and compatibility. The continued research and investment in new technologies has the potential to significantly benefit the treatment of life-threatening emergency injuries, both on the battlefield and in the civilian sector. In this review, we discuss military blood-management practices and military-specific uses of individual blood components, as well as describe and analyze several artificial blood products that could be options for future battlefield use.

Disruption of Biofilm by Bacteriophages in Clinically Relevant Settings.

INTRODUCTION: Antibiotic-resistant bacteria are a growing threat to civilian and military health today. Although infections were once easily treatable by antibiotics and wound cleaning, the frequent mutation of bacteria has created strains impermeable to antibiotics and physical attack. Bacteria further their pathogenicity because of their ability to form biofilms on wounds, medical devices, and implant surfaces. Methods for treating biofilms in clinical settings are limited, and when formed by antibiotic-resistant bacteria, can generate chronic infections that are recalcitrant to available therapies. Bacteriophages are natural viral predators of bacteria, and their ability to rapidly destroy their host has led to increased attention in potential phage therapy applications. MATERIALS AND METHODS: The present article sought to address a knowledge gap in the available literature pertaining to the usage of bacteriophage in clinically relevant settings and the resolution of infections particular to military concerns. PRISMA guidelines were followed for a systematic review of available literature that met the criteria for analysis and inclusion. The research completed for this review article originated from the U.S. Military Academy's library "Scout" search engine, which complies results from 254 available databases (including PubMed, Google Scholar, and SciFinder). The search criteria included original studies that employed bacteriophage use against biofilms, as well as successful phage therapy strategies for combating chronic bacterial infections. We specifically explored the use of bacteriophage against antibiotic- and treatment-resistant bacteria. RESULTS: A total of 80 studies were identified that met the inclusion criteria following PRISMA guidelines. The application of bacteriophage has been demonstrated to robustly disrupt biofilm growth in wounds and on implant surfaces. When traditional therapies have failed to disrupt biofilms and chronic infections, a combination of these treatments with phage has proven to be effective, often leading to complete wound healing without reinfection. CONCLUSIONS: This review article examines the available literature where bacteriophages have been utilized to treat biofilms in clinically relevant settings. Specific attention is paid to biofilms on implant medical devices, biofilms formed on wounds, and clinical outcomes, where phage treatment has been efficacious. In addition to the clinical benefit of phage therapies, the military relevance and treatment of combat-related infections is also examined. Phages offer the ability to expand available treatment options in austere environments with relatively low cost and effort, allowing the impacted warfighter to return to duty quicker and healthier.

The Epistemic Fallacy: Unintended Consequences of Empirically Treating (Clinically Diagnosed) Chronic Lyme Disease in a Soldier.

OBJECTIVE: We document a military patient presenting with a diffuse set of symptoms suggestive of chronic Lyme disease (CLD) and the subsequent empiric treatment and health complications arising therein. The lay medical community, spurred by the internet, has ascribed these diffuse symptoms to various illnesses including CLD without confirmatory serological evidence of any underlying disease. With a growing community of patient advocates, CLD has become an illness with broad and highly generalized list of clinical symptoms and an absence of agreed-upon confirmatory laboratory tests. Further complicating matters, diagnostic criteria and treatment protocols differ between the Infectious Diseases Society of America and the International Lyme and Associated Diseases Society guidelines. Clinicians also face serious challenges in diagnosing and treating patients who present with generalized symptoms and close to 50 diagnostic tests for Lyme disease available in North America. Further complicating the picture for military patients seeking medical confirmation of a disease and resolution of their symptoms, medical fitness boards use putative diagnoses as prima faciae evidence in disability. Here a military patient with a long list of complaints that defy any clear or easy diagnosis and treatment is discussed. However, these symptoms taken together with selectively summed notes in the medical record in the absence of convincing and clear laboratory confirmation are suggestive of CLD and its complications, but no resolution was ultimately reached. With the presumptive determination of a medical disability due to CLD by the medical board, the medical dismissal of this service member from active duty occurred.

Genomic surveillance of SARS-CoV-2 during the first year of the pandemic in the Bronx enabled clinical and epidemiological inference.

The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of SARS-CoV-2 genomes across the Bronx from March-October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of variants, we found that while some became 'endemic' to the Bronx, other, novel variants rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic. One sentence summary: Temporally and geographically resolved sequencing of SARS-CoV-2 genotypes enabled surveillance of novel genotypes, identification of endemic viral variants, and clinical inferences, in the first wave of the COVID-19 pandemic in the Bronx.

Genomic surveillance of SARS-CoV-2 during the first year of the pandemic in the Bronx enabled clinical and epidemiological inference.

The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of 104 SARS-CoV-2 genomes across the Bronx from March October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of mutations, we found that while some became 'endemic' to the Bronx, other, novel mutations rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic.

Detection of Potential Pathogenic Bacteria on the Surfaces of Female Urinary Diversion Devices Following a Short Duration Military Training Exercise.

BACKGROUND: Female Servicemembers are increasingly being incorporated into the combat arms and Special Operations communities. Female urinary diversion devices (FUDDs) have been used to facilitate urination in the austere environments that are encountered by Servicemembers. Importantly, the potential for the bacterial contamination of these devices has not been evaluated. The goals of this study were to determine whether microorganisms adhere to the surfaces of FUDDs in the field environment and to demonstrate the presence of potential pathogens on the used devices. MATERIALS AND METHODS: A total of 15 devices that were used in a comprehensive 18-24-hour military field exercise were tested for the presence of microorganisms. Briefly, each device was swabbed, and the swabs were used to inoculate blood agar plates to encourage bacterial growth. The resulting bacterial colonies were identified, and the surface topography of the devices was investigated with electron microscopy. RESULTS: Although microscopy revealed few surface features capable of facilitating bacterial attachment, several species were recovered. Significantly, a biofilm-forming strain of Proteus mirabilis (P. mirabilis) was detected on two of the devices. P. mirabilis is a mobile urinary pathogen that can potentially migrate from the surface of the device into the urinary tract of the user. CONCLUSION: Commercial FUDDs can support bacterial growth and harbor potential pathogens. Care should be taken to ensure that Servicemembers are aware of the importance of the proper care and cleaning of these devices in the field environment. To this end, standard operating procedures should be developed and distributed.

Escherichia coli as a Potential Reservoir of Antimicrobial Resistance Genes on the Island of O'ahu.

The problem of antimicrobial-resistant bacteria has not been adequately explored in the tropical island environment. To date, there has not been a systematic investigation into the prevalence and distribution of antimicrobial resistance determinants in the Hawaiian Islands. Urinary isolates are the most common bacterial pathogens encountered in the clinical laboratory. Therefore, the antimicrobial resistance determinant profiles of these organisms can serve as a sentinel of the overall antimicrobial resistance situation in a localized patient population. In this study, 82 clinical isolates of Escherichia coli derived from 82 distinct patients were collected at a large medical center on the island of O'ahu. Each isolate was evaluated for the presence of antimicrobial resistance genes using a microarray-based approach. A total of 36 antimicrobial resistance genes covering 10 classes of antimicrobial compounds were identified. Most isolates were found to harbor between 3 and 5 antimicrobial resistance genes. Only a few isolates were found to harbor more than 12 genes. Significantly, a high rate of phenotypic resistance to one of the first-line treatments for uncomplicated urinary tract infection (sulfamethoxazole) was identified. This phenotype was correlated to the presence of sulfonamides and trimethoprim resistance determinants. Since E. coli is one of the most encountered pathogens in the hospital environment, the presence of clinically relevant resistance determinants in isolates of this organism from a clinical setting on O'ahu is a significant finding that warrants further investigation.

A Longitudinal Evaluation of the Bacterial Pathogens Colonizing Chronic Non-Healing Wound Sites at a United States Military Treatment Facility in the Pacific Region.

PURPOSE: The biology of chronic wounds is complex and many factors act concurrently to impede healing progress. In this study, the dynamics of microflora changes and their antibiotic susceptibility patterns were evaluated longitudinally over 30 days using data from 28 patients with a total of 47 chronic lower extremity wounds. MATERIALS AND METHODS: In this study, colonized wound isolates were characterized using cultural, biochemical, and VITEK 2 methods. Antibiotic susceptibility patterns of the wound isolates were analyzed using various phenotypic assays. Furthermore, antimicrobial resistance patterns and the presence of mutations were evaluated by a genotypic assay, whole-genome sequencing (WGS). RESULTS: Staphylococcus aureus and Pseudomonas aeruginosa were found to be the most common strains at early time points, while members of Enterobacteriaceae were prevalent at later stages of infection. Antimicrobial resistance testing and whole-genome sequencing revealed that the molecular and phenotypic characteristics of the identified wound pathogens remained relatively stable throughout the study period. It was also noted that Enterobacter and Klebsiella species may serve as reservoirs for quinolone resistance in the Pacific region. CONCLUSION: Our observations showed that wounds were colonized with diverse bacteria and interestingly their numbers and/or types were changed over the course of infection. The rapid genetic changes that accompanied the first 4 weeks after presentation did not directly contribute to the development of antibiotic resistance. In addition, standard wound care procedures did not appear to select for resistant bacterial strains. Future efforts should focus on defining those genetic changes associated with the wound colonizing microorganisms that occur beyond 4 weeks.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Qualitative Immunoglobulin G Assays: The Value of Numeric Reporting.

CONTEXT.­: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) testing is used for serosurveillance and will be important to evaluate vaccination status. Given the urgency to release coronavirus disease 2019 (COVID-19) serology tests, most manufacturers have developed qualitative tests. OBJECTIVE.­: To evaluate clinical performance of 6 different SARS-CoV-2 IgG assays and their quantitative results to better elucidate the clinical role of serology testing in COVID-19. DESIGN.­: Six SARS-CoV-2 IgG assays were tested using remnant specimens from 190 patients. Sensitivity and specificity were evaluated for each assay with the current manufacturer's cutoff and a lower cutoff. A numeric result analysis and discrepancy analysis were performed. RESULTS.­: Specificity was higher than 93% for all assays, and sensitivity was higher than 80% for all assays (≥7 days post-polymerase chain reaction testing). Inpatients with more severe disease had higher numeric values compared with health care workers with mild or moderate disease. Several discrepant serology results were those just below the manufacturers' cutoff. CONCLUSIONS.­: Severe acute respiratory syndrome coronavirus 2 IgG antibody testing can aid in the diagnosis of COVID-19, especially with negative polymerase chain reaction. Quantitative COVID-19 IgG results are important to better understand the immunologic response and disease course of this novel virus and to assess immunity as part of future vaccination programs.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Treatment of severe COVID-19 with convalescent plasma in Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as a treatment for coronavirus disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200 mL of CCP with a spike protein IgG titer ≥ 1:2430 (median 1:47,385) within 72 hours of admission with propensity score-matched controls cared for at a medical center in the Bronx, between April 13 and May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroid use, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared with matched controls, CCP recipients less than 65 years had 4-fold lower risk of mortality and 4-fold lower risk of deterioration in oxygenation or mortality at day 28. For CCP recipients, pretransfusion spike protein IgG, IgM, and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients less than 65 years, but data from controlled trials are needed to validate this finding and establish the effect of aging on CCP efficacy.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Single-Dilution COVID-19 Antibody Test with Qualitative and Quantitative Readouts.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.

An evaluation of the impact of clinical bacterial isolates on epithelial cell monolayer integrity by the electric Cell-Substrate Impedance Sensing (ECIS) method.

Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using the Electric Cell-Substrate Impedance Sensing (ECIS) system. ECIS is a morphological biosensor which records electrical properties of cell-covered microelectrodes in an AC circuit including impedance (ohm), resistance (ohm), and capacitance (µFarad). In the current study, fluctuations in the electrical properties of cell-covered microelectrodes reflect dynamic changes in cell morphology resulting from disrupted cell monolayers following exposure to bacteria. Using the ECIS system, real-time changes of cell morphology and disruption of monolayer integrity of cell-cultures in vitro were revealed for A549 cells infected with either Pseudomonas aeruginosa, ESBL Escherichia coli, Staphylococcus aureus (MRSA), or Enterococcus (VRE). We determined empirically that the optimal signal response was obtained for resistance (ohm) measurements at 4000 hertz. Following infection of A549 cells, the data revealed that Pseudomonas aeruginosa resulted in little change in microelectrode resistance (ohm @4 kHz) as compared to pathogen-free controls within the first 12 h. In contrast, E. coli, MRSA, and VRE caused significant changes in electrode resistance (ohm @4 kHz) values in the infected cells compared to controls over the first 5 h. Resistance (ohm @4 kHz) changes were also observed in cell monolayers infected with different bacterial concentrations for all isolates over 24 h. The highest concentration of bacteria caused the measured resistance (ohm @4 kHz) to drop faster than its' immediate lower concentration, suggesting a dose-dependent effect. Compared to live bacteria, cells exposed to heat-killed bacteria did not show significant changes in resistance (ohm @4 kHz) over 48 h post-exposure. Functionally, cytokine responses were different between cells treated with live and heat-killed bacteria. Of note, live bacteria induced IFNγ, IL-13, and IL-1ß production in A549 cells, whereas heat-killed bacteria induced IL-8 production suggesting a differential interaction with cells that could reveal the underlying causes of resistance (ohm @4 kHz) changes. Our findings indicate that ECIS provides a means to quantify, automate, and measure bacterial virulence, which may have broader implications governing the course of treatment compared to traditional methods alone.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.