Performance of Three Anti-SARS-CoV-2 Anti-S and One Anti-N Immunoassays for the Monitoring of Immune Status and Vaccine Response.

This study aimed to evaluate and compare the performance of three anti-S and one anti-N assays that were available to the project in detecting antibody levels after three commonly used SARS-CoV-2 vaccines (Pfizer, Moderna, and Johnson & Johnson). It also aimed to assess the association of age, sex, race, ethnicity, vaccine timing, and vaccine side effects on antibody levels in a cohort of 827 individuals. In September 2021, 698 vaccinated individuals donated blood samples as part of the Association for Diagnostics & Laboratory Medicine (ADLM) COVID-19 Immunity Study. These individuals also participated in a comprehensive survey covering demographic information, vaccination status, and associated side effects. Additionally, 305 age- and gender-matched samples were obtained from the ADLM 2015 sample bank as pre-COVID-19-negative samples. All these samples underwent antibody level analysis using three anti-S assays, namely Beckman Access SARS-CoV-2 IgG (Beckman assay), Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG (Ortho assay), Siemens ADVIA Centaur SARS-CoV-2 IgG (Siemens assay), and one anti-N antibody assay: Bio-Rad Platelia SARS-CoV-2 Total Ab assay (BioRad assay). A total of 827 samples (580 COVID-19 samples and 247 pre-COVID-19 samples) received results for all four assays and underwent further analysis. Beckman, Ortho, and Siemens anti-S assays showed an overall sensitivity of 99.5%, 97.6%, and 96.9%, and specificity of 90%, 100%, and 99.6%, respectively. All three assays indicated 100% sensitivity for individuals who received the Moderna vaccine and boosters, and over 99% sensitivity for the Pfizer vaccine. Sensitivities varied from 70.4% (Siemens), 81.5% (Ortho), and 96.3% (Beckman) for individuals who received the Johnson & Johnson vaccine. BioRad anti-N assays demonstrated 46.2% sensitivity and 99.25% specificity based on results from individuals with self-reported infection. The highest median anti-S antibody levels were measured in individuals who received the Moderna vaccine, followed by Pfizer and then Johnson & Johnson vaccines. Higher anti-S antibody levels were significantly associated with younger age and closer proximity to the last vaccine dose but were not associated with gender, race, or ethnicity. Participants with higher anti-S levels experienced significantly more side effects as well as more severe side effects (e.g., muscle pain, chills, fever, and moderate limitations) (p < 0.05). Anti-N antibody levels only indicated a significant correlation with headache. This study indicated performance variations among different anti-S assays, both among themselves and when analyzing individuals with different SARS-CoV-2 vaccines. Caution should be exercised when conducting large-scale studies to ensure that the same platform and/or assays are used for the most effective interpretation of the data.

Large-Scale Scientific Study Led by a Professional Organization during the COVID-19 Pandemic: Operations, Best Practices, and Lessons Learned.

In 2021, the Association for Diagnostics & Laboratory Medicine (ADLM) (formerly the American Association for Clinical Chemistry [AACC]) developed a scientific study that aimed to contribute to the understanding of SARS-CoV-2 immunity during the evolving course of the pandemic. This study was led by a group of expert member volunteers and resulted in survey data from 975 individuals and blood collection from 698 of those participants. This paper describes the formulation and execution of this large-scale scientific study, encompassing best practices and insights gained throughout the endeavor.

Robust Response of the Clinical Laboratory to the COVID-19 Pandemic despite Significant Challenges.

BACKGROUND: Clinical laboratories immediately provided rapid, reliable, and high-throughout diagnostic testing for COVID-19, which was an essential component in combating the pandemic. As the pandemic evolved, the clinical laboratory was faced with additional challenges. However, there are limited studies on the impact of the pandemic on the clinical laboratory over the past 3 years. METHODS: The American Association for Clinical Chemistry (AACC) sent 8 surveys over a 32-month time period to international clinical laboratory leadership asking questions about COVID-19 testing, supplies, staffing, and lessons learned. RESULTS: There were a total of 191 unique respondents: 133 laboratories in the US and 58 laboratories from 37 other countries participated. By May 2020, more than 70% of laboratories offered COVID-19 diagnostic testing with average turnaround times ranging from 1 to 24 h. Daily COVID-19 testing volumes peaked in January of 2022 at a median of 775 tests per day. Throughout the pandemic, supplies and staffing concerns increased. In most of the 8 surveys, 55% to 65% of laboratories reported they were unable to obtain supplies. Obtaining reagents and test kits was the most problematic. Staffing challenges continue to be a significant concern and most laboratories have struggled hiring testing personnel. CONCLUSIONS: Survey results were utilized to demonstrate the impact of the pandemic on the clinical laboratory community, and importantly, findings were presented to the White House Coronavirus Taskforce. Overall, the clinical laboratories had a robust response to the COVID-19 pandemic, and despite ongoing and evolving challenges, continue to provide rapid diagnostic testing.

T Cell Responses Correlate with Self-Reported Disease Severity and Neutralizing Antibody Responses Predict Protection against SARS-CoV-2 Breakthrough Infection.

OBJECTIVES: The objective of this prospective study was to investigate the role of adaptive immunity in response to SARS-CoV-2 vaccines. DESIGN AND METHODS: A cohort of 677 vaccinated individuals participated in a comprehensive survey of their vaccination status and associated side effects, and donated blood to evaluate their adaptive immune responses by neutralizing antibody (NAb) and T cell responses. The cohort then completed a follow-up survey to investigate the occurrence of breakthrough infections. RESULTS: NAb levels were the highest in participants vaccinated with Moderna, followed by Pfizer and Johnson & Johnson. NAb levels decreased with time after vaccination with Pfizer and Johnson & Johnson. T cell responses showed no significant difference among the different vaccines and remained stable up to 10 months after the study period for all vaccine types. In multivariate analyses, NAb responses (<95 U/mL) predicted breakthrough infection, whereas previous infection, the type of vaccine, and T cell responses did not. T cell responses to viral epitopes (<0.120 IU/mL) showed a significant association with the self-reported severity of COVID-19 disease. CONCLUSION: This study provides evidence that NAb responses to SARS-CoV-2 vaccination correlate with protection against infection, whereas the T cell memory responses may contribute to protection against severe disease but not against infection.

Tolerance for three commonly administered COVID-19 vaccines by healthcare professionals.

Importance: Most healthcare institutions require employees to be vaccinated against SARS-CoV-2 and many also require at least one booster. Objective: We determine the impact of vaccine type, demographics, and health conditions on COVID-19 vaccine side effects in healthcare professionals. Design: A COVID-19 immunity study was performed at the 2021 American Association for Clinical Chemistry Annual Scientific meeting. As part of this study, a REDCap survey with cascading questions was administered from September 9, 2021 to October 20, 2021. General questions included participant demographics, past and present health conditions, smoking, exercise, and medications. COVID-19 specific questions asked about SARS-CoV-2 vaccine status and type, vaccine-associated side effects after each dose including any boosters, previous infection with COVID-19, diagnostic testing performed, and type and severity symptoms of COVID-19. Results: There were 975 participants (47.1% male, median age of 50 years) who completed the survey. Pfizer was the most commonly administered vaccine (56.4%) followed by Moderna (32.0%) and Johnson & Johnson (7.1%). There were no significant differences in vaccine type received by age, health conditions, smoking, exercise, or type or number of prescription medications. Side effects were reported more frequently after second dose (e.g., Moderna or Pfizer) (54.1%) or single/only dose of Johnson & Johnson (47.8%). Males were significantly more likely to report no side effects (p < 0.001), while females were significantly more likely to report injection site reactions (p < 0.001), fatigue (p < 0.001), headache (p < 0.001), muscle pain (p < 0.001), chills (p = 0.001), fever (p = 0.007), and nausea (p < 0.001). There was a significant upward trend in participants reporting no side effects with increasing age (p < 0.001). There were no significant trends in side effects among different races, ethnicities, health conditions, medications, smoking status or exercise. In multivariate logistic regressions analyses, the second dose of Moderna was associated with a significantly higher risk of side effects than both the second dose of Pfizer and the single dose of Johnson & Johnson. Conclusions and relevance: Younger people, females, and those receiving the second dose of Moderna had more COVID-19 vaccine side effects that per self-report led to moderate to severe limitations. As reported in other studies, the increase in side effects from Moderna may be explained by higher viral mRNA concentrations but be associated with additional protective immunity.

Heterogeneous phenotypes of Pten-null hepatocellular carcinoma in hepatitis B virus transgenic mice parallels liver lobule zonal gene expression patterns.

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The phenotypes of HCC are diverse, in part, due to mutations in distinct oncogenes and/or tumor suppressor genes. These genetic drivers of HCC development have generally been considered as major mediators of tumor heterogeneity. Using the liver-specific Pten-null HBV transgenic mouse model of chronic viral infection, a critical role for liver lobule zone-specific gene expression patterns in determining HCC phenotype and ß-catenin-dependent HBV biosynthesis is demonstrated. These observations suggest that the position of the hepatocyte within the liver lobule, and hence its intrinsic gene expression pattern at the time of cellular transformation, make critical contributions to the properties of the resulting liver tumor. These results may explain why therapies targeting pathways modulated by specific identified tumor driver genes display variable treatment efficacy.

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment.

Sirtuin enzymes depend on NAD+ to catalyze protein deacetylation. Therefore, the lowering of NAD+ during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD+-depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD+-limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms.

Role of peroxisome proliferator-activated receptor gamma coactivator 1alpha in AKT/PKB-mediated inhibition of hepatitis B virus biosynthesis.

Hepatitis B virus (HBV) transcription and replication are essentially restricted to hepatocytes because liver-enriched transcription factors govern viral RNA synthesis. The level of transcription from the HBV promoters depends on both the transcription factors binding to these regulatory sequence elements and their ability to recruit coactivators capable of mediating assembly of the transcription preinitiation complex containing RNA polymerase II. Nuclear receptors are a primary determinant of HBV pregenomic RNA synthesis and, hence, viral replication. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) enhances the activity of nuclear receptors and, consequently, HBV biosynthesis. PGC1α is also an important target of signal transduction pathways involved in hepatic glucose and lipid homeostasis, suggesting that this coactivator may have an important role in modulating HBV biosynthesis under various physiological conditions. Consistent with this suggestion, v-akt murine thymoma viral oncogene homolog/protein kinase B (AKT/PKB) is shown to modulate PGC1α activity and, hence, HBV transcription and replication in a cell line-specific manner. In addition, AKT can modulate HBV replication in some but not all cell lines at a posttranscriptional step in the viral life cycle. These observations demonstrate that growth and nutritional signals have the capacity to influence viral production, but the magnitude of these effects will depend on the precise cellular context in which they occur.

Distinct regulation of hepatitis B virus biosynthesis by peroxisome proliferator-activated receptor gamma coactivator 1alpha and small heterodimer partner in human hepatoma cell lines.

The human hepatoma cell lines HepG2 and Huh7 have been used extensively to study hepatitis B virus (HBV) transcription and replication. Both cell lines support transcription of the 3.5-kb viral pregenomic RNA and subsequent viral DNA synthesis by reverse transcription. The effects of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1alpha) and corepressor small heterodimer partner (SHP) on HBV transcription and replication mediated by nuclear receptors were examined in the context of individual nuclear receptors in nonhepatoma cells and in hepatoma cells in an attempt to determine the relative contribution of the various nuclear receptors to viral biosynthesis in the hepatoma cells. PGC1alpha and SHP modulated viral biosynthesis differently in the human hepatoma cell lines HepG2 and Huh7, indicating distinct modes of transcriptional regulation. Consistent with this suggestion, it appears that retinoid X receptor alpha/farnesoid X receptor alpha and liver receptor homolog 1 or estrogen-related receptor beta (ERRbeta) may contribute to the majority of the viral replication observed in HepG2 cells, whereas ERRalpha and ERRgamma are probably responsible for the majority of viral biosynthesis in Huh7 cells. Therefore, this approach indicates that the transcriptional regulation of HBV biosynthesis in HepG2 and Huh7 cells is primarily controlled by different transcription factors.

Peroxisome proliferator-activated receptor gamma Coactivator 1alpha and small heterodimer partner differentially regulate nuclear receptor-dependent hepatitis B virus biosynthesis.

Hepatitis B virus (HBV) biosynthesis involves the transcription of the 3.5-kb viral pregenomic RNA, followed by its reverse transcription into viral DNA. Consequently, the modulation of viral transcription influences the level of virus production. Nuclear receptors are the only transcription factors known to support viral pregenomic RNA transcription and replication. The coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1alpha) and corepressor small heterodimer partner (SHP) have central roles in regulating energy homeostasis in the liver by modulating the transcriptional activities of nuclear receptors. Therefore, the effect of PGC1alpha and SHP on HBV transcription and replication mediated by nuclear receptors was examined in the context of individual nuclear receptors in nonhepatoma cells and in hepatoma cells. This analysis indicated that viral replication mediated by hepatocyte nuclear factor 4alpha, retinoid X receptor alpha (RXRalpha) plus peroxisome proliferator-activated receptor alpha (PPARalpha), and estrogen-related receptor (ERR) displayed differential sensitivity to PGC1alpha activation and SHP inhibition. The effects of PGC1alpha and SHP on viral biosynthesis in the human hepatoma cell line Huh7 were similar to those observed in the nonhepatoma cells expressing ERRalpha and ERRgamma. This suggests that these nuclear receptors, potentially in combination with RXRalpha plus PPARalpha, may have a major role in governing HBV transcription and replication in this cell line. Additionally, this functional approach may help to distinguish the transcription factors in various liver cells governing viral biosynthesis under a variety of physiologically relevant conditions.