Transcriptional Modulation of Hepatitis B Virus Through Multiple Regulatory Pathways

Hepatitis B Virus (HBV) is a human pathogen that chronically infects approximately 400 million people worldwide. Although a vaccine exists to protect against HBV infection, there is no effective treatment for chronic carriers of this disease. The objective of this research is to understand the in vivo mechanisms through which HBV is regulated. We have established the capability of the transcriptional coactivator, peroxisome proliferator-activated receptor- γ coactivator 1α (PGC1α), to enhance viral biosynthesis by interacting with nuclear receptors required for recruitment of the transcriptional machinery necessary for viral RNA biosynthesis. While PGC1α has been demonstrated to be a powerful factor in enhancing viral biosynthesis in cell culture, modest results in vivo exemplify the significant challenges in demonstrating its specificity. The coactivators PGC1β, CBP, SRC1, and GRIP are also capable of enhancing viral biosynthesis, suggesting that compensation at the level of transcriptional coactivation exists, and that PGC1α is but one member in a multifactorial network influencing HBV biosynthesis. These specific coactivators can enhance nuclear receptor-mediated HBV gene transcription as well as gene activation by Fox transcription factors, presenting a previously unknown role for Fox transcription factors in HBV biosynthesis. It is likely that these transcription factors may be differentially expressed throughout the lifespan of a chronically infected patient, allowing the virus to persist through changing metabolic states. We also demonstrate that Akt/PKB (v-akt murine thymoma viral oncogene homolog/protein kinase B) is capable of regulating HBV biosynthesis through both transcriptional and post-transcriptional stages of the viral life cycle. This supports the hypothesis that HBV is metabolically regulated by signal transduction pathways in the liver. Akt activity is dependent on the insulin/PI3K/PDK1/Akt signal transduction pathway which is activated by growth/feeding conditions and inhibited during stress/starvation. This is consistent with previous work that has shown that caloric restriction has enhanced HBV biosynthesis, whereas loss of HBV has occurred during hepatocyte proliferation and tumor formation. Chronic HBV infection is associated with the development of liver disease and hepatocellular carcinoma. Developing the liver-specific PTEN-null HBV transgenic mouse, which produces constitutive Akt, allows the examination of the role of Akt in vivo. The aged PTEN-null mouse progressively develops hepatocellular carcinoma, which is often associated with Akt activation resulting from either growth factor stimulation or mutations within the Akt signal transduction pathway. HBV nucleocapsid protein expression and DNA replication are quite limited in tumors but relatively robust in adjacent nontumor liver tissue. Surprisingly, HBV 3.5kb RNA transcript levels are the same in both tumor and nontumor tissue. This suggests that secondary signaling events in the tumor post-transcriptionally inhibit viral biosynthesis. While viral biosynthesis has been previously observed to be limited in tumors, this study demonstrates that only distinct stages of the viral lifecycle are inhibited. Overall, this work provides insight into how HBV can adapt to various nutritional states and metabolic stresses. The role of metabolic signaling pathways, including the PI3K/Akt signaling pathway, bridges the metabolic state of the hepatocyte with HBV biosynthesis. Ultimately, our studies promote a more detailed mechanistic understanding of how chronic HBV persists in the liver and may identify novel targets for the future development of therapeutic agents

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

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

ImportanceMost healthcare institutions require employees to be vaccinated against SARS-CoV-2 and many also require at least one booster.ObjectiveWe determine the impact of vaccine type, demographics, and health conditions on COVID-19 vaccine side effects in healthcare professionals.DesignA 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.ResultsThere 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 relevanceYounger 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

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

ObjectivesThe objective of this prospective study was to investigate the role of adaptive immunity in response to SARS-CoV-2 vaccines.Design and methodsA 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.ResultsNAb 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.ConclusionThis 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

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