Introduction:Understanding factors that shape immune responses in wild animals is critical to predicting population resilience and long-term persistence. Immune function modifies the survival of individuals facing infectious disease, trauma, and environmental stressors, yet remains understudied. An individual's immune response is shaped not only by current and historic pathogen exposures but is mediated by both individual (e.g. host genetics, metabolic plane, age, and sex) and population-level (e.g. population size, density, and connectivity) factors. Bighorn sheep (Ovis canadensis, bighorn) occupy populations of varying sizes, nested within larger metapopulations, creating a hierarchical structure. This organization provides a useful framework to understand how immune parameters vary across individual, population, and metapopulation levels. Unfortunately, measurement of immune parameters in ungulates is limited. Methods:To address this limitation, we measured 18 immunologic traits across 581 wild bighorn to evaluate this toolkit's ability to detect immunological differences between individuals, populations, and metapopulations. Results:Most immunological phenotypes illustrated significant variation at the metapopulation level and individual level. Our assays revealed immune phenotypic variation consistent with two main axes of segregation-one that distinguished tradeoffs in bighorn innate versus adaptive immune responses, and another reflecting alternative inflammatory states, defined by distinct cytokine patterns. Bighorn age and sex also mediated immune response patterns. Conclusions:Our immunological toolkit sets the stage to further clarify landscape-level immunological variation in wild ungulate populations and provides a template for deploying integrative eco-immunological tools in any natural population to further understand wildlife health.

Both major species of elephants Loxodonta africana and Elephas maximus can live for more than 50 years in the wild and are listed as endangered species. Elephants have a unique leukocyte profile with approximately 50% of an individual’s leukocytes resembling monocytes. Such a high number of monocytes is more than other any other mammalian species, though there is debate as to the true identity of these cells. Why such high levels of monocytes are present and their function are unknown. Here we used single cell RNAseq to identify leukocyte populations from a captive Asian Elephant. Transcript sequences were compared to the African elephant genome, which is more highly annotated than the Asian elephant. Progressive uMAP analysis identified clusters of cells which corresponded to T cells, B cells, granulocytes, and monocytes. Importantly, the proportion of analyzed cells closely resembled the CBC counts from the same animal, indicating that our analysis could correctly distinguish cell types. We further restricted single cell transcriptome analysis of cells from myeloid origin, and, following exclusion of granulocytes, found that the remaining monocytes could be divided into four to six distinct subsets. Monocyte subsets were further analyzed by examining transcripts related to immune functions. These data confirm that elephants do in fact have a large population of monocytes in peripheral blood and will be useful in directing future studies to understand their function. Veterinary and Comparative Immunology (VET)

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development.

Antigenic peptides presented by MHC class I molecules at the surface of the cell are often derived from proteins degraded by the ubiquitin-proteasome system. Here we examined the relationship between protein ubiquitination and peptide presentation by chemically inhibiting the action of Cullin-Ring-Ligases (CRLs), a large family of ubiquitin ligases, and determining the fate of peptides presented from proteins targeted for ubiquitination and degradation by CRLs. Hela cells expressing secreted HLA-A*11:01 molecules were treated with MLN4924, an inhibitor of NEDDylation, which is essential for CRL functional activity. Peptides were isolated from secreted HLA molecules and identified by mass spectrometry. Approximately 2/3rd of source-proteins presented was largely unchanged following MLN4924 treatment, and, interestingly, changes to the identity of the immunopeptidome could not be explained by alterations to the cellular transcriptome. More than half of the peptides derived from substrates of CRLs were still presented after treatment with MLN4924. Western blot analysis demonstrated that numerous CRL substrates were spared from degradation following MLN4924 treatment. These data indicate that despite inhibiting ubiquitination of many proteins targeted for degradation, peptides are still liberated for MHC class I antigen presentation, perhaps due to alternative degradation pathways or defective forms of the protein degraded independently of their canonical ubiquitin ligase. NIH R01AI130059 Antigen and Dendritic Cell Processing, Presentation, and Biology (AGDC)

Supranutritional Se supplementation may improve immune responses in beef cattle. Immunity is compromised in beef cattle during the periparturient period. This study aims to determine the best time during pregnancy to supplement beef cows with Se-yeast to optimize humoral immunity at parturition. Multiparous, black Angus and Angus cross cows (n = 79) were used in the study. All cows had ad libitum access to a mineral supplement containing 120 mg/kg Se (US FDA regulations) from Na selenite. In addition, all cows except controls (CTR) received Se supplementation of 105 mg Se/week from Se-yeast boluses administered once weekly during their specific treatment trimester of gestation (TR1, TR2, or TR3) for 13 weeks. This dosage was supranutritional equaling 5 × the upper range of US FDA Se administration regulations. Blood was collected at parturition from all cows. Laboratory analyses studied to assess humoral immunity included measuring IBR, BVD types 1 and 2, PI3, and BRSV serum neutralization titers post vaccination, assessing total IgM and antigen-specific IgM concentrations, and determining complement-mediated bacterial killing percentages. Statistical analyses were performed using GraphPad Prism and SAS 9.4. Supranutritional Se-yeast supplementation increased whole-blood (WB) Se concentrations regardless of trimester of supplementation (all P < 0.0001). Supplementation during TR2 and TR3 was more effective in increasing WB-Se concentrations at parturition than during TR1 or CTR (all P < 0.0001). TR2 cows had higher serum neutralization titers for BRSV compared with CRT cows (P = 0.03). Total serum IgM and Vibrio coralliilyticus–specific IgM concentrations were highly correlated (r = 0.78; P < 0.0001). Compared with CTR cows, TR1, TR2, and TR3 cows had similar total IgM concentrations (all P ≥ 0.19) and similar Vibrio coralliilyticus–specific IgM concentrations (all P ≥ 0.47). Complement-mediated bacterial killing percentages were greater in TR2 and TR3 cows (> 99.6

Optimal veterinary care of managed elephant populations is vital due to the continued decline of wild populations. Appropriate health monitoring and accurate disease diagnosis include hematologic evaluation. Elephant hematology is distinctive in that elephants have high percentages of monocytes in health. Elephant monocytes also have unusual morphology, a feature shared with manatees and rock hyraxes. Manual white blood cell counting is used for elephant hematology, as analyzers are generally inaccurate. The aims of this study were to evaluate basic cell isolation and functional testing protocols for use in elephant monocyte research, and to test several available antibodies via flow cytometry for use in elephant monocyte identification. Peripheral blood samples from five Asian elephants (Elephas maximus) were used. Methods for monocyte isolation and evaluation of phagocytic function were established. Putative lymphocyte and monocyte populations were identified using a scatter on flow cytometry. Antibodies against CD11b, CD11c, CD14, and ionized calcium-binding adapter molecule 1 (IBA1) were tested, with IBA1 showing the highest apparent diagnostic utility in labeling monocytes. Combined flow cytometric scatter and IBA1 positivity appear to identify Asian elephant monocytes. These data provide a methodologic basis for further investigation into elephant monocyte function and immune response to infection.

Habitat fragmentation is an important driver of biodiversity loss and can be remediated through management actions aimed at maintenance of natural connectivity in metapopulations. Connectivity may protect populations from infectious diseases by preserving immunogenetic diversity and disease resistance. However, connectivity could exacerbate the risk of infectious disease spread across vulnerable populations. We tracked the spread of a novel strain of Mycoplasma ovipneumoniae in a metapopulation of desert bighorn sheep Ovis canadensis nelsoni in the Mojave Desert to investigate how variation in connectivity among populations influenced disease outcomes. M. ovipneumoniae was detected throughout the metapopulation, indicating that the relative isolation of many of these populations did not protect them from pathogen invasion. However, we show that connectivity among bighorn sheep populations was correlated with higher immunogenetic diversity, a protective immune response and lower disease prevalence. Variation in protective immunity predicted infection risk in individual bighorn sheep and was associated with heterozygosity at genetic loci linked to adaptive and innate immune signalling. Together, these findings may indicate that population connectivity maintains immunogenetic diversity in bighorn sheep populations in this system and has direct effects on immune responses in individual bighorn sheep and their susceptibility to infection by a deadly pathogen. Our study suggests that the genetic benefits of population connectivity could outweigh the risk of infectious disease spread and supports conservation management that maintains natural connectivity in metapopulations.

The emergence of immunotherapy for the treatment of human cancers has heralded a new era in oncology, one that is making its way into the veterinary clinic. As the immune system of many animal species commonly seen by veterinarians is similar to humans, there is great hope for the translation of human therapies into veterinary oncology. The simplest approach for veterinarians would be to adopt existing reagents that have been developed for human medicine, due to the potential of reduced cost and the time it takes to develop a new drug. However, this strategy may not always prove to be effective and safe with regard to certain drug platforms. Here, we review current therapeutic strategies that could exploit human reagents in veterinary medicine and also those therapies which may prove detrimental when human-specific biological molecules are used in veterinary oncology. In keeping with a One Health framework, we also discuss the potential use of single-domain antibodies (sdAbs) derived from camelid species (also known as Nanobodies™) for therapies targeting multiple veterinary animal patients without the need for species-specific reformulation. Such reagents would not only benefit the health of our veterinary species but could also guide human medicine by studying the effects of outbred animals that develop spontaneous tumors, a more relevant model of human diseases compared to traditional laboratory rodent models.

Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)–proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell–expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product–derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation. Key Points NEDD8 fusion to a model protein results in proteasome and autophagosome degradation. NEDD8 fusion is less efficient than ubiquitin fusion for peptide presentation. DRiP Ag presentation is diminished by MLN4924 treatment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the most severe pandemic in a century. The virus gains access to host cells when the viral Spike protein (S-protein) binds to the host cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interaction with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often these cells only transiently express ACE2 proteins and levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bi-cistronic vector with an easy to quantify reporter protein to normalize ACE2 expression. We found that both binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus is proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of reporter protein, Thy1.1. We also compared different ACE2 orthologs which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein while human ACE2 was the highest level detected and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variant's ability to potentially infect different animals.IMPORTANCE:SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here we describe a method to generate cells stably expressing equivalent levels of different ACE2 orthologs, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both binding of the viral Spike protein receptor binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to ACE2 levels at the cell surface. Adaptation of this method will allow for the creation of a library of stable transfected cells expressing equivalent levels of different vertebrate ACE2 orthologs which can be repeatedly used for identifying vertebrate species which may be susceptible to infection with SARS-CoV-2 and its many variants.

Neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) is a ubiquitin-like protein (UBL) whose canonical function involves binding to, and thus, activating Cullin–Ring finger Ligases (CRLs), one of the largest family of ubiquitin ligases in the eukaryotic cell. However, in recent years, several non-canonical protein substrates of NEDD8 have been identified. Here we attempt to review the recent literature regarding non-canonical NEDDylation of substrates with a particular focus on how the covalent modification of NEDD8 alters the protein substrate. Like much in the study of ubiquitin and UBLs, there are no clear and all-encompassing explanations to satisfy the textbooks. In some instances, NEDD8 modification appears to alter the substrates localization, particularly during times of stress. NEDDylation may also have conflicting impacts upon a protein’s stability: some reports indicate NEDDylation may protect against degradation whereas others show NEDDylation can promote degradation. We also examine how many of the in vitro studies measuring non-canonical NEDDylation were conducted and compare those conditions to those which may occur in vivo, such as cancer progression. It is likely that the conditions used to study non-canonical NEDDylation are similar to some types of cancers, such as glioblastoma, colon and rectal cancers, and lung adenocarcinomas. Although the full outcomes of non-canonical NEDDylation remain unknown, our review of the literature suggests that researchers keep an open mind to the situations where this modification occurs and determine the functional impacts of NEDD8-modification to the specific substrates which they study.

Abstract Desert bighorn sheep (Ovis canadensis nelsoni) are a charismatic ungulate native to western North America that live in mountainous regions. In the desert of the southwest United States, populations of bighorn sheep can be isolated by both natural and anthropogenic barriers. Isolation limits gene flow, exacerbates loss of genetic diversity to drift, and thus could affect immune responses to infectious agents. We sought to develop a relatively rapid method for determining MHC class I gene diversity in individual sheep. Both DNA and RNA were isolated from leukocytes collected from 154 adult bighorn sheep from across different mountain ranges in Southern California. Previously identified primers from domestic sheep were used to amplify exons 2 and 3 from OMHC I genes while appending adapters for PacBio circular consensus sequencing. We successfully identified over 40 unique MHC class I sequences expressed by bighorn sheep. Using a combination of phylogenic tree analysis and mapping MHC sequences to the domestic sheep genome, we detect at least 4 unique classical MHC class I regions. Comparison of results using either genomic DNA or mRNA (cDNA) as a template for analysis suggest that certain MHC alleles are expressed at different levels within leukocytes, while many potential pseudogenes were identified when genomic DNA was used as a template. Our data suggests using messenger RNA (cDNA) as a template to identify MHC class I diversity is superior to genomic DNA. Finally, we found clear patterns of MHC class I genotypes differentiated by population, which suggests that both natural and anthropogenic-induced population fragmentation can limit the diversity of MHC genes within a given population.

The ability to modulate direct MHC class I (MHC I) Ag presentation is a desirable goal for the treatment of a variety of conditions, including autoimmune diseases, chronic viral infections, and cancers. It is therefore necessary to understand how changes in the cellular environment alter the cells' ability to present peptides to T cells. The unfolded protein response (UPR) is a signaling pathway activated by the presence of excess unfolded proteins in the endoplasmic reticulum. Previous studies have indicated that chemical induction of the UPR decreases direct MHC I Ag presentation, but the precise mechanisms are unknown. In this study, we used a variety of small molecule modulators of different UPR signaling pathways to query which UPR signaling pathways can alter Ag presentation in both murine and human cells. When signaling through the PERK pathway, and subsequent eIF2α phosphorylation, was blocked by treatment with GSK2656157, MHC I Ag presentation remain unchanged, whereas treatment with salubrinal, which has the opposite effect of GSK2656157, decreases both Ag presentation and overall cell-surface MHC I levels. Treatment with 4μ8C, an inhibitor of the IRE1α UPR activation pathway that blocks splicing of Xbp1 mRNA, also diminished MHC I Ag presentation. However, 4μ8C treatment unexpectedly led to an increase in eIF2α phosphorylation in addition to blocking IRE1α signaling. Given that salubrinal and 4μ8C lead to eIF2α phosphorylation and similar decreases in Ag presentation, we conclude that UPR signaling through PERK, leading to eIF2α phosphorylation, results in a modest decrease in direct MHC I Ag presentation.

While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

MHC class I molecules present peptide antigens derived from proteins synthesized and degraded within the antigen presenting cell. An understanding the protein degradation pathways that contribute to peptide generation is necessary for developing next generation immunotherapies. We recently discovered that conjugation of the ubiquitin-like molecule NEDD8 to the N-terminus of a protein can result in the rapid degradation of the target protein by either the ubiquitin-proteasome system (UPS) and the autophagy pathway. To determine if protein NEDDylation increased peptide presentation, we fused NEDD8 to the N-terminus of a cytosolic form of ovalbumin (OVA) and measured presentation of the SIINFEKL peptide via the murine MHC class I molecule Kb using a monoclonal antibody specific for the Kb-SIINFEKL complex. Like other constructs we have characterized, NEDDylated OVA was rapidly degraded by the UPS and autophagy pathways. While proteasome inhibition prevented presentation of SIINFEKL peptides, inhibiting autophagy did not affect antigen presentation. We then compared presentation of NEDDylated OVA to ubiquitin conjugated OVA and found that ubiquitin conjugation was far more effective at generating antigenic peptides. These data demonstrate that rapid degradation per se of an antigenic protein is not sufficient to enhance antigen presentation but rather the precise manner in which a protein is degraded governs the efficiency of peptide generation. Our findings are consistent with the idea that protein degradation is organized in some way to optimize generation the immunopeptidome for immunosurveillance.

Diversity of MHC class I alleles within animal populations is necessary to prevent pathogen escape from adaptive immune responses. However, demonstrating this phenomenon is difficult especially in wild animal species where the effect of pathogen escape is more profound than in laboratory animal models. Desert bighorn sheep (Ovis canadensis nelsoni) are a charismatic megafauna native to the southwest of North America which are threatened by a number of infectious diseases and, more recently, paranasal tumors. We sought to develop a relatively inexpensive and rapid way to identify the diversity of MHC class I genes and alleles within individual bighorn sheep. Both DNA and RNA were isolated from leukocytes collected from 154 adult bighorn sheep from across different mountain ranges in Southern California. Previously identified primers from domestic sheep were used to amplify exons 2 and 3 from OMHC I genes while appending adapters for PacBio circular consensus sequencing. PCR amplicons from individual animals were then subjected to a second round of PCR to append index sequences to allow assigning of individual sequences to unique animals. We successfully identified over 40 unique MHC class I sequences expressed by bighorn sheep. Between 2 and 5 loci were amplified in each individual animal. Analysis of gDNA revealed several pseudogenes and other DNA sequences which were not transcribed. Finally, we demonstrate that particular DNA sequences are found within particular populations, suggesting that as bighorn populations fragment, there is a loss of MHC class I diversity.

Two genotypes of the intestinal parasite Ceratonova shasta infect Oncorhynchus mykiss: genotype 0 results in a chronic infection with low mortality while genotype IIR causes disease with high mortality. We determined parasite load and the relative expression of six immune factors (IgT, IgM, IL-6, IL-8, IL-10, IFNG) in fish infected with either genotype over 29 days post-exposure. In genotype IIR infections the host responded with upregulation of inflammatory and regulatory cytokines. In contrast, genotype 0 infection did not elicit an inflammatory response and expression of IFNG and IL-10 was lower. Antibody expression was upregulated in both infections but appeared to have limited efficacy in the virulent genotype IIR infections. Histologically, in genotype 0 infections the parasite migrated through the tissue layers causing inflammation but minimal damage to the mucosal epithelium, which contrasts with the severe pathology found in genotype IIR infections.