Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

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.

Third COVID-19 vaccine dose boosts neutralizing antibodies in poor responders.

Background: While evaluating COVID-19 vaccine responses using a rapid neutralizing antibody (NAb) test, we observed that 25% of mRNA vaccine recipients did not neutralize >50%. We termed this group "vaccine poor responders" (VPRs). The objective of this study was to determine if individuals who neutralized <50% would remain VPRs, or if a third dose would elicit high levels of NAbs. Methods: 269 healthy individuals ranging in age from 19 to 80 (Average age = 51; 165 females and 104 males) who received either BNT162b2 (Pfizer) or mRNA-1273 (Moderna) vaccines were evaluated. NAb levels were measured: (i) 2-4 weeks after a second vaccine dose, (ii) 2-4 months after the second dose, (iii) within 1-2 weeks prior to a third dose and (iv) 2-4 weeks after a third mRNA vaccine dose. Results: Analysis of vaccine recipients reveals that 25% did not neutralize above 50% (Median neutralization = 21%, titers <1:80) within a month after their second dose. Twenty-three of these VPRs obtained a third dose of either BNT162b2 or mRNA-1273 vaccine 1-8 months (average = 5 months) after their second dose. Within a month after their third dose, VPRs show an average 5.4-fold increase in NAb levels (range: 46-99%). Conclusions: The results suggest that VPRs are not permanently poor responders; they can generate high NAb levels with an additional vaccine dose. Although it is not known what levels of NAbs protect from infection or disease, those in high-risk professions may wish to keep peripheral NAb levels high, limiting infection, and potential transmission.

Development of a rapid point-of-care test that measures neutralizing antibodies to SARS-CoV-2.

BACKGROUND: After receiving a COVID-19 vaccine, most recipients want to know if they are protected from infection and for how long. Since neutralizing antibodies are a correlate of protection, we developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies from a drop of blood. The LFA is based on the principle that neutralizing antibodies block binding of the receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). METHODS: The ability of the LFA was assessed to correctly measure neutralization of sera, plasma or whole blood from patients with COVID-19 using SARS-CoV-2 microneutralization assays. We also determined if the LFA distinguished patients with seasonal respiratory viruses from patients with COVID-19. To demonstrate the usefulness of the LFA, we tested previously infected and non-infected COVID-19 vaccine recipients at baseline and after first and second vaccine doses. RESULTS: The LFA compared favorably with SARS-CoV-2 microneutralization assays with an area under the ROC curve of 98%. Sera obtained from patients with seasonal coronaviruses did not show neutralizing activity in the LFA. After a single mRNA vaccine dose, 87% of previously infected individuals demonstrated high levels of neutralizing antibodies. However, if individuals were not previously infected, only 24% demonstrated high levels of neutralizing antibodies after one vaccine dose. A second dose boosted neutralizing antibody levels just 8% higher in previously infected individuals, but over 63% higher in non-infected individuals. CONCLUSIONS: A rapid, semi-quantitative, highly portable and inexpensive neutralizing antibody test might be useful for monitoring rise and fall in vaccine-induced neutralizing antibodies to COVID-19.

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.

Cationic gold microparticles for biolistic delivery of nucleic acids.

Here we report preparation and properties of positively charged gold microparticles, and their use for biolistic DNA delivery. Micron-sized gold microparticles were modified by building self-assembling polyethylenimine monolayers on their surfaces, which enabled their electrostatic interaction with negatively charged molecules such as DNA. One milligram of the surface-modified microparticles was able to bind directly to up to 3.5 microg of DNA, exceeding the 1 microg/mg limit of the conventional protocols. The binding showed no apparent dependency on DNA purity, size, or conformation. The interaction occurred over a broad range of pH values and salt concentrations, and was stable throughout the standard protocol for biolistic cartridge preparation. At the standard 1 microg dose, biological activity of the DNA biolistically delivered on the charge-modified gold was 25% higher than that delivered on conventional microparticles. Loading the charge-modified gold with more DNA stimulated proportionally higher gene expression. The charge-modified gold can be also used for delivery of small biological molecules such as siRNA. Tissue culture cells biolistically transfected with a LUC+ specific siRNA showed 80% reduction of Luc expression relative to those cells transfected with an irrelevant siRNA. Along with its superior properties as a DNA delivery vehicle, charge-modified gold offers a unique opportunity to deliver various DNA formulations in addition to traditional naked DNA.

Label-free impedimetric detection of glycan-lectin interactions.

A compact biosensor for a label-free, rapid (<80 s) detection of glycan-lectin interactions using ac impedance measurements was developed for the first time. A galactose-binding peanut agglutinin (PNA) and sialic acid-binding Sambucus nigra agglutinin (SNA) were covalently surface-immobilized on the layered Cu/Ni/Au printed circuit board (PCB) electrodes. Samples of artificial and natural glycoconjugates consisting of (1) gold glyconanoparticles encapsulated with approximately 90-100 copies of TF-antigen disaccharide Galalpha1-3GalNAc (TF-AuNP), (2) asialofetuin (ASF) containing both LacNAc (Galbeta1-4GlcNAc) and TF-antigen, and (3) fetuin (FET), the sialylated glycoform of ASF. The samples were run separately on PNA- and SNA-immobilized PCB electrodes. Our results indicate that TF-AuNP could be rapidly and reliably detected up to 1 pg/mL (13 fM) concentration on PNA electrode but, as expected, yielded no response on the SNA electrode. ASF and FET glycocoproteins were unambiguously detectable up to 10 pg/mL (150 fM) on PNA and SNA electrodes, respectively. Moreover, the technique allowed us to observe glyco-microheterogeneity of FET as well as establish the presence of two isoforms of SNA lectin, SNA-I and SNA-II, in one of the vendor's formulations. Further elaboration of the described technology into novel electrochemically driven lectin arrays may find applications in diagnosis of cancer and other diseases with multiple glycobiomarkers or as a rapid low-cost bioanalytical tool for glycoproteome analyses.