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1. A dried blood spot‐based method to measure levels of tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a marker of bone resorption

   https://doi.org/10.1002/ajhb.23240  

   Eick, Geeta N. ; Devlin, Maureen J. ; Cepon‐Robins, Tara J. ; Kowal, Paul ; Sugiyama, Lawrence S. ; Snodgrass, J. Josh ( March 2019 , American Journal of Human Biology)

   A number of basic questions about bone biology have not been answered, including population differences in bone turnover. In part, this stems from the lack of validated minimally invasive biomarker techniques to measure bone formation and resorption in field‐based population‐level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a well‐defined biomarker of bone resorption and osteoclast number.

   We adapted a commercially available enzyme‐linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACP‐5b levels in serum and plasma for use with DBS. We used a rigorous process of assay modification and validation, including the use of a matched set of 189 adult plasma, fDBS, and venous DBS (vDBS) samples; parameters evaluated included precision, reliability, and analyte stability.

   Plasma and DBS TRACP‐5b concentrations showed a linear relationship. There were no systematic differences in TRACP‐5b levels in fDBS and vDBS, indicating no significant differences in TRACP‐5b distribution between capillary and venous blood. Parallelism and spike‐and‐recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACP‐5b levels from DBS using the validated kit. Intra‐ and interassay CVs were 5.0% and 12.1%, respectively. DBS samples should preferably be stored frozen but controlled room temperature storage for up to a month may be acceptable.

   This DBS‐based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.

    

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2. Hydrogel‐Based Slow Release of a Receptor‐Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS‐CoV‐2

   https://doi.org/10.1002/adma.202104362  

   Gale, Emily C. ; Powell, Abigail E. ; Roth, Gillie A. ; Meany, Emily L. ; Yan, Jerry ; Ou, Ben S. ; Grosskopf, Abigail K. ; Adamska, Julia ; Picece, Vittoria C. T. M. ; d'Aquino, Andrea I. ; et al ( October 2021 , Advanced Materials)

   The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID‐19. The receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically‐relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime‐boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer‐nanoparticle (PNP) hydrogel elicited potent anti‐RBD and anti‐spike antibody titers, providing broader protection against SARS‐CoV‐2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically‐relevant adjuvant systems. Notably, a SARS‐CoV‐2 spike‐pseudotyped lentivirus neutralization assay revealed that hydrogel‐based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

    

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3. Using Split Luminescent Biosensors for SARS‐CoV‐2 Antibody Detection in Serum, Plasma, and Blood Samples

   https://doi.org/10.1002/cpz1.521  

   Elledge, Susanna K. ; Eigl, Ian ; Phelps, Maira ; McClinton, Khayla ; Zhou, Xin X. ; Leung, Kevin K. ; Tato, Cristina M. ; Wells, James A. ( October 2022 , Current Protocols)

   Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID‐19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme‐linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS‐CoV‐2 virus to enable luminescent‐based detection of spike‐ or nucleocapsid‐binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low‐/medium‐throughput scale using plate‐based assays, high‐throughput scale using robotics, and point‐of‐care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid‐handling robotics and in a point‐of‐care setting using a handheld, battery‐powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point‐of‐care, nonlaboratory settings. © 2022 Wiley Periodicals LLC.

   Basic Protocol: SARS‐CoV‐2 antibody detection using the split‐luciferase assay on a medium‐throughput scale with a laboratory luminometer

   Alternate Protocol 1: High‐throughput‐based protocol for SARS‐CoV‐2 antibody detection using a robotic platform

   Alternate Protocol 2: Point‐of‐care‐based protocol for SARS‐CoV‐2 antibody detection using a handheld luminometer

   Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days

    

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4. Neutralizing antibodies targeting the SARS‐CoV‐2 receptor binding domain isolated from a naïve human antibody library

   https://doi.org/10.1002/pro.4044  

   Bell, Benjamin N. ; Powell, Abigail E. ; Rodriguez, Carlos ; Cochran, Jennifer R. ; Kim, Peter S. ( February 2021 , Protein Science)

   Infection with SARS‐CoV‐2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient‐derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS‐CoV‐2 neutralizing antibodies. Here, we used a yeast surface‐display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin‐converting enzyme 2 (ACE2), the human receptor for SARS‐CoV‐2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS‐CoV‐2 spike‐pseudotyped lentivirus with IC₅₀values as low as 60 ng/ml in vitro. Using a biolayer interferometry‐based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID‐19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS‐CoV‐2 RBD.

    

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5. Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development

   https://doi.org/10.3390/ijms241914622  

   Zak, Andrew J. ; Hoang, Trang ; Yee, Christine M. ; Rizvi, Syed M. ; Prabhu, Ponnandy ; Wen, Fei ( September 2023 , International Journal of Molecular Sciences)

   Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04–0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72–144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

    

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