Arrayed Imaging Reflectometry monitoring of anti-viral antibody production throughout vaccination and breakthrough Covid-19.

Immune responses to COVID-19 infection and vaccination are individual and varied. There is a need to understand the timeline of vaccination efficacy against current and yet to be discovered viral mutations. Assessing immunity to SARS-CoV-2 in the context of immunity to other respiratory viruses is also valuable. Here we demonstrate the capability of a fully automated prototype Arrayed Imaging Reflectometry system to perform reliable longitudinal serology against a 34-plex respiratory array. The array contains antigens for respiratory syncytial virus, seasonal influenza, common human coronaviruses, MERS, SARS-CoV-1, and SARS-CoV-2. AIR measures a change in reflectivity due to the binding of serum antibodies to the antigens on the array. Samples were collected from convalescent COVID-19 donors and individuals vaccinated with a two-dose mRNA vaccine regimen. Vaccinated samples were collected prior to the first dose, one week after the first dose, one week after the second dose, and monthly thereafter. Information following booster dose and/or breakthrough infection is included for a subset of subjects. Longitudinal samples of vaccinated individuals demonstrate a rise and fall of SARS-CoV-2 spike antibodies in agreement with general knowledge of the adaptive immune response and other studies. Linear Regression analysis was performed to understand the relationship between antibodies binding to different antigens on the array. Our analysis identified strong correlations between closely related influenza virus strains as well as correlations between SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. A small test of using diluted whole blood from a fingerstick provided clean arrays with antibody binding comparable to serum. Potential applications include assessing immunity in the context of exposure to multiple respiratory viruses, clinical serology, population monitoring to facilitate public health recommendations, and vaccine development against new viruses and virus mutations.

Formation and Expansion of Memory B Cells against Coronavirus in Acutely Infected COVID-19 Individuals.

Infection with the ß-coronavirus SARS-CoV-2 typically generates strong virus-specific antibody production. Antibody responses against novel features of SARS-CoV-2 proteins require naïve B cell activation, but there is a growing appreciation that conserved regions are recognized by pre-existing memory B cells (MBCs) generated by endemic coronaviruses. The current study investigated the role of pre-existing cross-reactive coronavirus memory in the antibody response to the viral spike (S) and nucleocapsid (N) proteins following SARS-CoV-2 infection. The breadth of reactivity of circulating antibodies, plasmablasts, and MBCs was analyzed. Acutely infected subjects generated strong IgG responses to the S protein, including the novel receptor binding domain, the conserved S2 region, and to the N protein. The response included reactivity to the S of endemic ß-coronaviruses and, interestingly, to the N of an endemic α-coronavirus. Both mild and severe infection expanded IgG MBC populations reactive to the S of SARS-CoV-2 and endemic ß-coronaviruses. Avidity of S-reactive IgG antibodies and MBCs increased after infection. Overall, findings indicate that the response to the S and N of SARS-CoV-2 involves pre-existing MBC activation and adaptation to novel features of the proteins, along with the potential of imprinting to shape the response to SARS-CoV-2 infection.

Monitoring Serum Spike Protein with Disposable Photonic Biosensors Following SARS-CoV-2 Vaccination.

While mRNA vaccines have been well-studied in vitro and in animals prior to their use in the human population during the Covid-19 pandemic, their exact mechanisms of inducing immunity are still being elucidated. The large-scale collection of data necessary to fully understand these mechanisms, and their variability across heterogeneous populations, requires rapid diagnostic tests that accurately measure the various biomarkers involved in the immune response following vaccination. Recently, our lab developed a novel "Disposable Photonics" platform for rapid, label-free, scalable diagnostics that utilizes photonic ring resonator sensor chips combined with plastic micropillar cards able to provide passive microfluidic flow. Here, we demonstrate the utility of this system in confirming the presence of SARS-CoV-2 spike protein in the serum of recently vaccinated subjects, as well as tracking a post-vaccination rise in anti-SARS-CoV-2 antibodies. A maximum concentration in SARS-CoV-2 spike protein was detected one day after vaccination and was reduced below detectable levels within 10 days. This highlights the applicability of our rapid photonic sensor platform for acquiring the data necessary to understand vaccine mechanisms on a large scale, as well as individual patient responses to SARS-CoV-2 mRNA vaccines.

Targeting Ribosomal Frameshifting as an Antiviral Strategy: From HIV-1 to SARS-CoV-2.

Treatment of HIV-1 has largely involved targeting viral enzymes using a cocktail of inhibitors. However, resistance to these inhibitors and toxicity in the long term have pushed the field to identify new therapeutic targets. To that end, -1 programmed ribosomal frameshifting (-1 PRF) has gained attention as a potential node for therapeutic intervention. In this process, a ribosome moves one nucleotide backward in the course of translating a mRNA, revealing a new reading frame for protein synthesis. In HIV-1, -1 PRF allows the virus to regulate the ratios of enzymatic and structural proteins as needed for correct viral particle assembly. Two RNA structural elements are central to -1 PRF in HIV: a slippery sequence and a highly conserved stable hairpin called the HIV-1 frameshifting stimulatory signal (FSS). Dysregulation of -1 PRF is deleterious for the virus. Thus, -1 PRF is an attractive target for new antiviral development. It is important to note that HIV-1 is not the only virus exploiting -1 PRF for regulating production of its proteins. Coronaviruses, including the COVID-19 pandemic virus SARS-CoV-2, also rely on -1 PRF. In SARS-CoV-2 and other coronaviruses, -1 PRF is required for synthesis of RNA-dependent RNA polymerase and several other nonstructural proteins. Coronaviruses employ a more complex RNA structural element for regulating -1 PRF called a pseudoknot. The purpose of this Account is primarily to review the development of molecules targeting HIV-1 -1 PRF. These approaches are case studies illustrating how the entire pipeline from screening to the generation of high-affinity leads might be implemented. We consider both target-based and function-based screening, with a particular focus on our group's approach beginning with a resin-bound dynamic combinatorial library (RBDCL) screen. We then used rational design approaches to optimize binding affinity, selectivity, and cellular bioavailability. Our tactic is, to the best of our knowledge, the only study resulting in compounds that bind specifically to the HIV-1 FSS RNA and reduce infectivity of laboratory and drug-resistant strains of HIV-1 in human cells. Lessons learned from strategies targeting -1 PRF HIV-1 might provide solutions in the development of antivirals in areas of unmet medical need. This includes the development of new frameshift-altering therapies for SARS-CoV-2, approaches to which are very recently beginning to appear.

Disposable photonics for cost-effective clinical bioassays: application to COVID-19 antibody testing.

Decades of research have shown that biosensors using photonic circuits fabricated using CMOS processes can be highly sensitive, selective, and quantitative. Unfortunately, the cost of these sensors combined with the complexity of sample handling systems has limited the use of such sensors in clinical diagnostics. We present a new "disposable photonics" sensor platform in which rice-sized (1 × 4 mm) silicon nitride ring resonator sensor chips are paired with plastic micropillar fluidic cards for sample handling and optical detection. We demonstrate the utility of the platform in the context of detecting human antibodies to SARS-CoV-2, both in convalescent COVID-19 patients and for subjects undergoing vaccination. Given its ability to provide quantitative data on human samples in a simple, low-cost single-use format, we anticipate that this platform will find broad utility in clinical diagnostics for a broad range of assays.

Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients.

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.