Longitudinal Comparison of Neutralizing Antibody Responses to COVID-19 mRNA Vaccines after Second and Third Doses.

COVID-19 mRNA vaccines protect against severe disease and hospitalization. Neutralizing antibodies (NAbs) are a first-line defense mechanism, but protective NAb responses are variable. Currently, NAb testing is not widely available. This study employed a lateral flow assay for monitoring NAb levels postvaccination and natural infection, using a finger-stick drop of blood. We report longitudinal NAb data from BNT162b2 (Pfizer) and mRNA-1273 (Moderna) recipients after second and third doses. Results demonstrate a third dose of mRNA vaccine elicits higher and more durable NAb titers than the second dose, independent of manufacturer, sex, and age. Our analyses also revealed that vaccinated individuals could be categorized as strong, moderate, and poorly neutralizing responders. After the second dose, 34% of subjects were classified as strong responders, compared to 79% after the third dose. The final months of this study coincided with the emergence of the SARS-CoV-2 Omicron variant and symptomatic breakthrough infections within our study population. Lastly, we show that NAb levels sufficient for protection from symptomatic infection with early SARS-CoV-2 variants were not protective against Omicron infection and disease. This work highlights the need for accessible vaccine response monitoring for use in healthcare, such that individuals, particularly those in vulnerable populations, can make informed vaccination decisions.

SARS-CoV-2 Serologic Assays Dependent on Dual-Antigen Binding Demonstrate Diverging Kinetics Relative to Other Antibody Detection Methods.

Longitudinal studies assessing durability of the anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) humoral immune response have generated conflicting results. This has been proposed to be due to differences in patient populations, the lack of standardized methodologies, and the use of assays that measure distinct aspects of the humoral response. SARS-CoV-2 antibodies were serially measured in sera from a cohort of 44 well-characterized convalescent plasma donors over 120 days post-COVID-19 symptom onset, utilizing eight assays, which varied according to antigen source, the detected antibody isotype, and the activity measured (i.e., binding, blocking, or neutralizing). While the majority of assays demonstrated a gradual decline in antibody titers over the course of 120 days, the two electrochemiluminescence immunoassay Roche assays (Roche Diagnostics Elecsys anti-SARS-CoV-2 [qualitative, nucleocapsid based] and Roche Diagnostics Elecsys anti-SARS-CoV-2 S [semiquantitative, spike based]), which utilize dual-antigen binding for antibody detection, demonstrated stable and/or increasing antibody titers over the study period. This study is among the first to assess longitudinal, rather than cross-sectional, SARS-CoV-2 antibody profiles among convalescent COVID-19 patients, primarily using commercially available serologic assays with Food and Drug Administration emergency use authorization. We show that SARS-CoV-2 antibody detection is dependent on the serologic method used, which has implications for future assay utilization and clinical value.

High-throughput platform for rapid deployment of antimicrobial agents.

A new approach to conducting bacterial binding assays by using an addressable high density random sequence peptide microarray is described. When bacterial binding is carried out in the presence of a competing excess of corresponding bacterial lipopolysaccharide (LPS), most of the observed bacterial binding is inhibited, suggesting that LPS is the major target of the bacterial binding peptides. Importantly, the amino acid composition of the selected peptides closely resembles the composition of natural antimicrobial peptides. Conjugation of selected peptides to polyvalent nanoparticle scaffold yields constructs that show potent antibacterial agglutination activities. The system is general enough to potentially create antimicrobial agents to virtually any pathogen.

A method for unobtrusive labeling of lipopolysaccharides with quantum dots.

Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of innate immune system, yet mechanisms of their action and, in particular, the role of the glycans remain elusive. Efficient noninvasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here, we describe a new method for labeling LPS and other lipoglycans with luminescent quantum dots (QDots). The labeling is achieved by the partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS-QDot conjugates is demonstrated by labeling of mouse monocytes. This simple method will find broad applicability in studies concerned with visualization of LPS biodistribution and identification of LPS-binding agents.

Self-assembled micronanoplexes for improved biolistic delivery of nucleic acids.

A new method for biolistic delivery of nucleic acids using a combination of cationic micro- and nanoparticles is reported. The new method is simpler to perform than the conventional calcium/spermidine-based formulations and shows 11-fold improved nucleic acid binding capacity and dose-dependent performance both for in vitro and in vivo applications relative to either the conventional preparation or our recently reported direct cationic microparticle method. These features may enable higher throughput gene delivery and genetic immunization programs and open new venues for the biolistic delivery method.

Peptide microarrays for carbohydrate recognition.

An application of high density random sequence peptide microarrays for rapid and reliable identification of artificial carbohydrate receptors is reported.

Bacterial glycoprofiling by using random sequence peptide microarrays.

Current analytical methods have been slow in addressing the growing need for glyco-analysis. A new generation of more empirical high-throughput (HTP) tools is needed to aid the advance of this important field. To this end, we have developed a new HTP screening platform for identification of surface-immobilized peptides that specifically bind O-antigenic glycans of bacterial lipopolysaccharides (LPS). This method involves screening of random sequence peptide libraries in addressable high-density microarray format with the newly developed luminescent LPS-quantum dot micelles. Screening of LPS fractions from O111:B4 and O55:B5 serotypes of E. coli on a microarray consisting of 10,000 20-mer peptide features revealed minor differences, while comparison of LPS from E. coli O111:B4 and P. aeruginosa produced sets of highly specific peptides. Peptides strongly binding to the E. coli LPS were highly enriched in aromatic and cationic amino acids, and most of these inhibited growth of E. coli. Flow cytometry and isothermal titration calorimetry (ITC) experiments showed that some of these peptides bind LPS in-solution with a K(d) of 1.75 microM. Peptide selections against P. aeruginosa were largely composed of hydrogen-bond forming amino acids in accordance with dramatic compositional differences in O-antigenic glycans in E. coli and P. aeruginosa. While the main value of this approach lies in the ability to rapidly differentiate bacterial and possibly other complex glycans, the peptides discovered here can potentially be used off-array as antiendotoxic and antimicrobial lead compounds, and on-array/on-bead as diagnostic and affinity reagents.

Facile labeling of lipoglycans with quantum dots.

Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of the innate immune system, yet mechanisms of their action and in particular the role of glycans remain elusive. Efficient non-invasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here we report a new method for labeling LPS and other lipoglycans with luminescent quantum dots. The labeling is achieved by partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS-Qdot conjugates is demonstrated by the labeling of mouse monocytes. This simple method should find broad applicability in studies concerned with visualization of LPS biodistribution and identification of LPS binding agents.