Amyloidogenic propensity of self-assembling peptides and their adjuvant potential for use as DNA vaccines.

De novo designed peptides that self-assemble into cross-ß rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant- and inflammation-free vaccines. However, they share structural and morphological properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their successful translation. Here, we comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-ß peptide KFE8, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE8 backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE8 is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE8 fibrils do not cross-seed amyloid formation of α-syn in mammalian cells compared to α-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE32-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8+T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-ß-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attractive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles. STATEMENT OF SIGNIFICANCE: Biomaterials comprised of self-assembling peptides hold great promise for the development of new vaccines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-ß rich fibrils that are structurally similar to amyloid species implicated in disease. Here, we comprehensively study the properties of these biomaterials. We demonstrate that they have distinct properties from pathological proteins. They are non-toxic and do not trigger amyloidogenesis. Vaccination of these materials in mice elicited a robust immune response. Most excitingly, our work suggests that this platform could be used to develop DNA-based vaccines, which have few storage requirements. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification.

Anticoagulant rodenticides are associated with increased stress and reduced body condition of avian scavengers in the Pacific Northwest.

Anticoagulant rodenticides (AR) have been used globally to manage commensal rodents for decades. However their application has also resulted in primary, secondary, and tertiary poisoning in wildlife. Widespread exposure to ARs (primarily second generation ARs; SGARs) in raptors and avian scavengers has triggered considerable conservation concern over their potential effects on populations. To identify risk to extant raptor and avian scavenger populations in Oregon and potential future risk to the California condor (Gymnogyps californianus) flock recently established in northern California, we assessed AR exposure and physiological responses in two avian scavenger species (common ravens [Corvus corax] and turkey vultures [Cathartes aura]) throughout Oregon between 2013 and 2019. AR exposure was widespread with 51% (35/68) of common ravens and 86% (63/73) of turkey vultures containing AR residues. The more acutely toxic SGAR brodifacoum was present in 83% and 90% of AR exposed common ravens and turkey vultures. The odds of AR exposure in common ravens were 4.7-fold higher along the coastal region compared to interior Oregon. For common ravens and turkey vultures that were exposed to ARs, respectively, 54% and 56% had concentrations that exceeded the 5% probability of toxicosis (>20 ng/g ww; Thomas et al., 2011), and 20% and 5% exceeded the 20% probability of toxicosis (>80 ng/g ww; Thomas et al., 2011). Common ravens exhibited a physiological response to AR exposure with fecal corticosterone metabolites increasing with sum ARs (ΣAR) concentrations. Both female common raven and turkey vultures' body condition was negatively correlated with increasing ΣAR concentrations. Our results suggest avian scavengers in Oregon are experiencing extensive AR exposure and the newly established population of California condors in northern California may experience similar AR exposure if they feed in southern Oregon. Understanding the sources of ARs across the landscape is an important first step in reducing or eliminating AR exposure in avian scavengers.

Small-Mammal Shooting as a Conduit for Lead Exposure in Avian Scavengers.

Lead (Pb) exposure is a widespread wildlife conservation threat. Although commonly associated with Pb-based ammunition from big-game hunting, small mammals (e.g., ground squirrels) shot for recreational or pest-management purposes represent a potentially important Pb vector in agricultural regions. We measured the responses of avian scavengers to pest-shooting events and examined their Pb exposure through consumption of shot mammals. There were 3.4-fold more avian scavengers at shooting fields relative to those at fields with no recent shooting, and avian scavengers spent 1.8-fold more time feeding after recent shooting events. We isotopically labeled shot ground squirrels in the field with an enriched 15N isotope tracer; 6% of avian scavengers sampled within a 39 km radius reflected this tracer in their blood. However, 33% of the avian scavengers within the average foraging dispersal distance of nests (0.6-3.7 km) were labeled, demonstrating the importance of these shooting fields as a source of food for birds nesting in close proximity. Additionally, Pb concentrations in 48% of avian scavengers exceeded subclinical poisoning benchmarks for sensitive species (0.03-0.20 µg/g w/w), and those birds exhibited reduced δ-aminolevulinic acid dehydratase activity, indicating a biochemical effect of Pb. The use of shooting to manage small mammal pests is a common practice globally. Efforts that can reduce the use of Pb-based ammunition may lessen the negative physiological effects of Pb exposure on avian scavengers.

Correlates of immune defenses in golden eagle nestlings.

An individual's investment in constitutive immune defenses depends on both intrinsic and extrinsic factors. We examined how Leucocytozoon parasite presence, body condition (scaled mass), heterophil-to-lymphocyte (H:L) ratio, sex, and age affected immune defenses in golden eagle (Aquila chrysaetos) nestlings from three regions: California, Oregon, and Idaho. We quantified hemolytic-complement activity and bacterial killing ability, two measures of constitutive immunity. Body condition and age did not affect immune defenses. However, eagles with lower H:L ratios had lower complement activity, corroborating other findings that animals in better condition sometimes invest less in constitutive immunity. In addition, eagles with Leucocytozoon infections had higher concentrations of circulating complement proteins but not elevated opsonizing proteins for all microbes, and eagles from Oregon had significantly higher constitutive immunity than those from California or Idaho. We posit that Oregon eagles might have elevated immune defenses because they are exposed to more endoparasites than eagles from California or Idaho, and our results confirmed that the OR region has the highest rate of Leucocytozoon infections. Our study examined immune function in a free-living, long-lived raptor species, whereas most avian ecoimmunological research focuses on passerines. Thus, our research informs a broad perspective regarding the evolutionary and environmental pressures on immune function in birds.

Changes in productivity and contaminants in bald eagles nesting along the lower Columbia River, USA.

Previous studies documented poor productivity of bald eagles (Haliaeetus leucocephalus) in the lower Columbia River (LCR), USA, and elevated p,p'-dichlorodiphenyldichloroethylene (DDE), polychlorinated biphenyls (PCBs), dioxins, and furans in eagle eggs. From 1994 to 1995, we collected partially incubated eggs at 19 of 43 occupied territories along the LCR and compared productivity and egg contaminants to values obtained in the mid-1980s. We found higher productivity at new nesting sites along the river, yet productivity at 23 older breeding territories remained low and was not different (p = 0.713) between studies. Eggshell thickness at older territories had not improved (p = 0.404), and eggshells averaged 11% thinner than shells measured before dichlorodiphenyltrichloroethane use. Decreases in DDE (p = 0.022) and total PCBs (p = 0.0004) in eggs from older breeding areas occurred between study periods. Productivity was not correlated to contaminants, but DDE, PCBs, and dioxin-like chemicals exceeded estimated no-effect values. Some dioxin-like contaminants in eggs were correlated to nest location, with highest concentrations occurring toward the river's mouth where productivity was lowest. Although total productivity increased due to the success of new nesting pairs in the region, egg contaminants remain high enough to impair reproduction at older territories and, over time, may alter productivity of new pairs nesting near the river's mouth.