Offspring affected with in utero Zika virus infection retain molecular footprints in the bone marrow and blood cells.

Congenital virus infections, for example cytomegalovirus and rubella virus infections, commonly affect the central nervous and hematological systems in fetuses and offspring. However, interactions between emerging congenital Zika virus and hematological system-bone marrow and blood-in fetuses and offspring are mainly unknown. Our overall goal was to determine whether silent in utero Zika virus infection can cause functional and molecular footprints in the bone marrow and blood of fetuses and offspring. We specifically focused on silent fetal infection because delayed health complications in initially asymptomatic offspring were previously demonstrated in animal and human studies. Using a well-established porcine model for Zika virus infection and a set of cellular and molecular experimental tools, we showed that silent in utero infection causes multi-organ inflammation in fetuses and local inflammation in the fetal bone marrow. In utero infection also caused footprints in the offspring bone marrow and PBMCs. These findings should be considered in a broader clinical context because of growing concerns about health sequelae in cohorts of children affected with congenital Zika virus infection in the Americas. Understanding virus-induced molecular mechanisms of immune activation and inflammation in fetuses may provide targets for early in utero interventions. Also, identifying early biomarkers of in utero-acquired immunopathology in offspring may help to alleviate long-term sequelae.

Plasma Cytokines and Birth Weight as Biomarkers of Vaccine-Induced Humoral Responses in Piglets.

Failure to mount an effective immune response to vaccination leaves individuals at risk for infection and can compromise herd immunity. Vaccine unresponsiveness can range from poor responses "low responders" to a failure to seroconvert "non-responders." Biomarkers of vaccine unresponsiveness, particularly those measured at the time of vaccination, could facilitate more strategic vaccination programs. We previously reported that pro-inflammatory cytokine signaling within peripheral blood mononuclear cells, elevated plasma interferon-gamma (IFNγ), and low birth weight correlated with vaccine-induced serum IgG titers in piglets that were below the threshold of detectable seroconversion (vaccine non-responders). These observations suggested that plasma IFNγ concentration and birth weight might serve as pre-vaccination biomarkers of vaccine unresponsiveness. To test this hypothesis, piglets (n = 67) from a different production facility were vaccinated with the same commercial Mycoplasma hyopneumoniae bacterin (RespiSure-One) to determine if there was a consistent and significant association between vaccine-induced serum IgG titers and either plasma cytokine concentrations or birth weight. All piglets seroconverted following vaccination with significantly less variability in vaccine-induced serum IgG titers than observed in the previous vaccine trial. Piglets exhibited highly variable birth weights and plasma cytokine concentrations prior to vaccination, but there were no significant associations (p > 0.05) between these variables and vaccine-induced serum IgG titers. There were significant (p < 0.001) differences in plasma IFNγ concentrations among individual litters (n = 6), and plasma IFNγ concentrations decreased in all pigs from birth to 63-days of age. One of the six litters (n = 11 piglets) exhibited significantly elevated plasma IFNγ concentrations during the first 3 weeks of life (p < 0.001) and at the time of vaccination (p < 0.01). This litter, however, had similar vaccine-induced serum IgG titers when compared to the other piglets in this study. Collectively the two studies indicate that while plasma cytokines and birth weight can be associated with vaccine non-responsiveness, their temporal and individual variation, as well as the complexity of the vaccine responsiveness phenotype, make them inconsistent biomarkers for predicting the less extreme phenotype of vaccine low responders.

Signaling differences in peripheral blood mononuclear cells of high and low vaccine responders prior to, and following, vaccination in piglets.

Individual variability in responses to vaccination can result in vaccinated subjects failing to develop a protective immune response. Vaccine non-responders can remain susceptible to infection and may compromise efforts to achieve herd immunity. Biomarkers of vaccine unresponsiveness could aid vaccine research and development as well as strategically improve vaccine administration programs. We previously vaccinated piglets (n = 117) against a commercial Mycoplasma hyopneumoniae vaccine (RespiSure-One) and observed in low vaccine responder piglets, as defined by serum IgG antibody titers, differential phosphorylation of peptides involved in pro-inflammatory cytokine signaling within peripheral blood mononuclear cells (PBMCs) prior to vaccination, elevated plasma interferon-gamma concentrations, and lower birth weight compared to high vaccine responder piglets. In the current study, we use kinome analysis to investigate signaling events within PBMCs collected from the same high and low vaccine responders at 2 and 6 days post-vaccination. Furthermore, we evaluate the use of inflammatory plasma cytokines, birthweight, and signaling events as biomarkers of vaccine unresponsiveness in a validation cohort of high and low vaccine responders. Differential phosphorylation events (FDR < 0.05) within PBMCs are established between high and low responders at the time of vaccination and at six days post-vaccination. A subset of these phosphorylation events were determined to be consistently differentially phosphorylated (p < 0.05) in the validation cohort of high and low vaccine responders. In contrast, there were no differences in birth weight (p > 0.5) and plasma IFNγ concentrations at the time of vaccination (p > 0.6) between high and low responders within the validation cohort. The results in this study suggest, at least within this study population, phosphorylation biomarkers are more robust predictors of vaccine responsiveness than other physiological markers.

High-resolution analysis of long-term serum antibodies in humans following convalescence of SARS-CoV-2 infection.

Long-term antibody responses to SARS-CoV-2 have focused on responses to full-length spike protein, specific domains within spike, or nucleoprotein. In this study, we used high-density peptide microarrays representing the complete proteome of SARS-CoV-2 to identify binding sites (epitopes) targeted by antibodies present in the blood of COVID-19 resolved cases at 5 months post-diagnosis. Compared to previous studies that evaluated epitope-specific responses early post-diagnosis (< 60 days), we found that epitope-specific responses to nucleoprotein and spike protein have contracted, and that responses to membrane protein have expanded. Although antibody titers to full-length spike and nucleoprotein remain steady over months, taken together our data suggest that the population of epitope-specific antibodies that contribute to this reactivity is dynamic and evolves over time. Further, the spike epitopes bound by polyclonal antibodies in COVID-19 convalescent serum samples aligned with known target sites that can neutralize viral activity suggesting that the maintenance of these antibodies might provide rapid serological immunity. Finally, the most dominant epitopes for membrane protein and spike showed high diagnostic accuracy providing novel biomarkers to refine blood-based antibody tests. This study provides new insights into the specific regions of SARS-CoV-2 targeted by serum antibodies long after infection.

Identification and Mechanistic Characterization of a Peptide Inhibitor of Glycogen Synthase Kinase (GSK3ß) Derived from the Disrupted in Schizophrenia 1 (DISC1) Protein.

Glycogen synthase kinase 3-beta (GSK3ß) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3ß have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3ß activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3ß in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3ß: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3ß binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3ß.

Kinome profiling of peripheral blood mononuclear cells collected prior to vaccination reveals biomarkers and potential mechanisms of vaccine unresponsiveness in pigs.

Inter-individual variance in host immune responses following vaccination can result in failure to develop protective immunity leaving individuals at risk for infection in addition to compromising herd immunity. While developing more efficacious vaccines is one strategy to mitigate this problem, predicting vaccine responsiveness prior to vaccination could inform which individuals require adjunct disease management strategies. To identify biomarkers of vaccine responsiveness, a cohort of pigs (n = 120) were vaccinated and pigs representing the high (n = 6; 90th percentile) and low (n = 6; 10th percentile) responders based on vaccine-specific antibody responses following vaccination were further analyzed. Kinase-mediated phosphorylation events within peripheral blood mononuclear cells collected prior to vaccination identified 53 differentially phosphorylated peptides when comparing low responders with high responders. Functional enrichment analysis revealed pro-inflammatory cytokine signaling pathways as dysregulated, and this was further substantiated by detection of higher (p < 0.01) concentrations of interferon-gamma in plasma of low responders compared to high responders prior to vaccination. In addition, low responder pigs with high plasma interferon-gamma showed lower (p < 0.01) birth weights than high responder pigs. These associations between vaccine responsiveness, cytokine signaling within peripheral immune cells, and body weight in pigs provide both evidence and insight into potential biomarkers for identifying low responders to vaccination.

From Beef to Bees: High-Throughput Kinome Analysis to Understand Host Responses of Livestock Species to Infectious Diseases and Industry-Associated Stress.

Within human health research, the remarkable utility of kinase inhibitors as therapeutics has motivated efforts to understand biology at the level of global cellular kinase activity (the kinome). In contrast, the diminished potential for using kinase inhibitors in food animals has dampened efforts to translate this research approach to livestock species. This, in our opinion, was a lost opportunity for livestock researchers given the unique potential of kinome analysis to offer insight into complex biology. To remedy this situation, our lab developed user-friendly, cost-effective approaches for kinome analysis that can be readily incorporated into most research programs but with a specific priority to enable the technology to livestock researchers. These contributions include the development of custom software programs for the creation of species-specific kinome arrays as well as comprehensive deconvolution and analysis of kinome array data. Presented in this review are examples of the application of kinome analysis to highlight the utility of the technology to further our understanding of two key complex biological events of priority to the livestock industry: host immune responses to infectious diseases and animal stress responses. These advances and examples of application aim to provide both mechanisms and motivation for researchers, particularly livestock researchers, to incorporate kinome analysis into their research programs.