Vector-Borne Disease in Wild Mammals Impacted by Urban Expansion and Climate Change.

Ecologies of zoonotic vector-borne diseases may shift with climate and land use change. As many urban-adapted mammals can host ectoparasites and pathogens of human and animal health concern, our goal was to compare patterns of arthropod-borne disease among medium-sized mammals across gradients of rural to urban landscapes in multiple regions of California. DNA of Anaplasma phagocytophilum was found in 1-5% of raccoons, coyotes, and San Joaquin kit foxes; Borrelia burgdorferi in one coyote, rickettsiae in two desert kit foxes, and Yersinia pestis in two coyotes. There was serological evidence of rickettsiae in 14-37% of coyotes, Virginia opossums, and foxes; and A. phagocytophilum in 6-40% of coyotes, raccoons, Virginia opossums, and foxes. Of six flea species, one Ctenocephalides felis from a raccoon was positive for Y. pestis, and Ct. felis and Pulex simulans fleas tested positive for Rickettsia felis and R. senegalensis. A Dermacentor similis tick off a San Joaquin kit fox was PCR-positive for A. phagocytophilum. There were three statistically significant risk factors: risk of A. phagocytophilum PCR-positivity was threefold greater in fall vs the other three seasons; hosts adjacent to urban areas had sevenfold increased A. phagocytophilum seropositivity compared with urban and rural areas; and there was a significant spatial cluster of rickettsiae within greater Los Angeles. Animals in areas where urban and rural habitats interconnect can serve as sentinels during times of change in disease risk.

Pathogenic Leptospira are widespread in the urban wildlife of southern California.

Leptospirosis, the most widespread zoonotic disease in the world, is broadly understudied in multi-host wildlife systems. Knowledge gaps regarding Leptospira circulation in wildlife, particularly in densely populated areas, contribute to frequent misdiagnoses in humans and domestic animals. We assessed Leptospira prevalence levels and risk factors in five target wildlife species across the greater Los Angeles region: striped skunks (Mephitis mephitis), raccoons (Procyon lotor), coyotes (Canis latrans), Virginia opossums (Didelphis virginiana), and fox squirrels (Sciurus niger). We sampled more than 960 individual animals, including over 700 from target species in the greater Los Angeles region, and an additional 266 sampled opportunistically from other California regions and species. In the five target species seroprevalences ranged from 5 to 60%, and infection prevalences ranged from 0.8 to 15.2% in all except fox squirrels (0%). Leptospira phylogenomics and patterns of serologic reactivity suggest that mainland terrestrial wildlife, particularly mesocarnivores, could be the source of repeated observed introductions of Leptospira into local marine and island ecosystems. Overall, we found evidence of widespread Leptospira exposure in wildlife across Los Angeles and surrounding regions. This indicates exposure risk for humans and domestic animals and highlights that this pathogen can circulate endemically in many wildlife species even in densely populated urban areas.

Landscape analyses using eDNA metabarcoding and Earth observation predict community biodiversity in California.

Ecosystems globally are under threat from ongoing anthropogenic environmental change. Effective conservation management requires more thorough biodiversity surveys that can reveal system-level patterns and that can be applied rapidly across space and time. Using modern ecological models and community science, we integrate environmental DNA and Earth observations to produce a time snapshot of regional biodiversity patterns and provide multi-scalar community-level characterization. We collected 278 samples in spring 2017 from coastal, shrub, and lowland forest sites in California, a complex ecosystem and biodiversity hotspot. We recovered 16,118 taxonomic entries from eDNA analyses and compiled associated traditional observations and environmental data to assess how well they predicted alpha, beta, and zeta diversity. We found that local habitat classification was diagnostic of community composition and distinct communities and organisms in different kingdoms are predicted by different environmental variables. Nonetheless, gradient forest models of 915 families recovered by eDNA analysis and using BIOCLIM variables, Sentinel-2 satellite data, human impact, and topographical features as predictors, explained 35% of the variance in community turnover. Elevation, sand percentage, and photosynthetic activities (NDVI32) were the top predictors. In addition to this signal of environmental filtering, we found a positive relationship between environmentally predicted families and their numbers of biotic interactions, suggesting environmental change could have a disproportionate effect on community networks. Together, these analyses show that coupling eDNA with environmental predictors including remote sensing data has capacity to test proposed Essential Biodiversity Variables and create new landscape biodiversity baselines that span the tree of life.

SARS-CoV-2: Cross-scale Insights from Ecology and Evolution.

Ecological and evolutionary processes govern the fitness, propagation, and interactions of organisms through space and time, and viruses are no exception. While coronavirus disease 2019 (COVID-19) research has primarily emphasized virological, clinical, and epidemiological perspectives, crucial aspects of the pandemic are fundamentally ecological or evolutionary. Here, we highlight five conceptual domains of ecology and evolution - invasion, consumer-resource interactions, spatial ecology, diversity, and adaptation - that illuminate (sometimes unexpectedly) the emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We describe the applications of these concepts across levels of biological organization and spatial scales, including within individual hosts, host populations, and multispecies communities. Together, these perspectives illustrate the integrative power of ecological and evolutionary ideas and highlight the benefits of interdisciplinary thinking for understanding emerging viruses.

Quantifying antibody kinetics and RNA detection during early-phase SARS-CoV-2 infection by time since symptom onset.

Understanding and mitigating SARS-CoV-2 transmission hinges on antibody and viral RNA data that inform exposure and shedding, but extensive variation in assays, study group demographics and laboratory protocols across published studies confounds inference of true biological patterns. Our meta-analysis leverages 3214 datapoints from 516 individuals in 21 studies to reveal that seroconversion of both IgG and IgM occurs around 12 days post-symptom onset (range 1-40), with extensive individual variation that is not significantly associated with disease severity. IgG and IgM detection probabilities increase from roughly 10% at symptom onset to 98-100% by day 22, after which IgM wanes while IgG remains reliably detectable. RNA detection probability decreases from roughly 90% to zero by day 30, and is highest in feces and lower respiratory tract samples. Our findings provide a coherent evidence base for interpreting clinical diagnostics, and for the mathematical models and serological surveys that underpin public health policies.

Estimating prevalence and test accuracy in disease ecology: How Bayesian latent class analysis can boost or bias imperfect test results.

Obtaining accurate estimates of disease prevalence is crucial for the monitoring and management of wildlife populations but can be difficult if different diagnostic tests yield conflicting results and if the accuracy of each diagnostic test is unknown. Bayesian latent class analysis (BLCA) modeling offers a potential solution, providing estimates of prevalence levels and diagnostic test accuracy under the realistic assumption that no diagnostic test is perfect.In typical applications of this approach, the specificity of one test is fixed at or close to 100%, allowing the model to simultaneously estimate the sensitivity and specificity of all other tests, in addition to infection prevalence. In wildlife systems, a test with near-perfect specificity is not always available, so we simulated data to investigate how decreasing this fixed specificity value affects the accuracy of model estimates.We used simulations to explore how the trade-off between diagnostic test specificity and sensitivity impacts prevalence estimates and found that directional biases depend on pathogen prevalence. Both the precision and accuracy of results depend on the sample size, the diagnostic tests used, and the true infection prevalence, so these factors should be considered when applying BLCA to estimate disease prevalence and diagnostic test accuracy in wildlife systems. A wildlife disease case study, focusing on leptospirosis in California sea lions, demonstrated the potential for Bayesian latent class methods to provide reliable estimates under real-world conditions.We delineate conditions under which BLCA improves upon the results from a single diagnostic across a range of prevalence levels and sample sizes, demonstrating when this method is preferable for disease ecologists working in a wide variety of pathogen systems.

Cancer targeting by TCR gene-engineered T cells directed against Kita-Kyushu Lung Cancer Antigen-1.

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83+ tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies.

A Phase II Study of Tumor-infiltrating Lymphocyte Therapy for Human Papillomavirus-associated Epithelial Cancers.

PURPOSE: Cellular therapy is an emerging cancer treatment modality, but its application to epithelial cancers has been limited. This clinical trial evaluated tumor-infiltrating lymphocyte (TIL) therapy for the treatment of patients with metastatic human papillomavirus (HPV)-associated carcinomas. PATIENTS AND METHODS: The trial was a phase II design with two cohorts, cervical cancers and noncervical cancers. Cell infusion was preceded by a lymphocyte-depleting conditioning regimen and followed by systemic high-dose aldesleukin. RESULTS: Objective tumor responses occurred in 5 of 18 (28%) patients in the cervical cancer cohort and 2 of 11 (18%) patients in the noncervical cancer cohort. Two of the responses in cervical cancer were complete and are ongoing 67 and 53 months after treatment. Responses in the noncervical cancer cohort were in anal cancer and oropharyngeal cancer. The HPV reactivity of the infused T cells correlated with clinical response. Peripheral blood repopulation with HPV-reactive T cells also correlated with clinical response. CONCLUSIONS: These findings support the concept that cellular therapy can mediate the regression of epithelial cancers, and they suggest the importance of predictive biomarkers and novel treatment platforms for more effective therapies.

Human Papillomavirus T-Cell Cross-reactivity in Cervical Cancer: Implications for Immunotherapy Clinical Trial Design.

Importance: Clinical trials are testing vaccines that target human papillomavirus 16 (HPV-16) oncoproteins for the treatment of cervical cancer regardless of the HPV type of the tumor. For patients with HPV-18-positive cancers, this strategy relies on cross-reactivity of HPV-16-reactive T cells against the HPV-18 oncoproteins. Objectives: To determine the prevalence of HPV-16 and HPV-18 metastatic cervical cancers in women enrolling in clinical trials at a US medical center and to assess whether HPV oncoprotein-targeting tumor-infiltrating lymphocytes (TILs) and T-cell receptors (TCRs) possess HPV-16/HPV-18 oncoprotein cross-reactivity. Design, Setting, and Participants: This study was conducted at the National Institutes of Health Clinical Center, a tertiary care research hospital in the United States. The HPV type of the tumors from 65 consecutive patients with cervical cancer who were evaluated for participation in clinical trials was determined by retrospective medical record review. Immunological assays testing HPV cross-reactivity were conducted on all available archived samples of oncoprotein-reactive TILs from HPV-positive tumors (n = 16) and on a library of previously identified TCRs (n = 10). Interventions: The HPV genotype of each patient's tumor was determined. The cross-reactivity of archived TILs and a library of TCRs was assessed. Main Outcomes and Measures: The main outcomes were the prevalence of each HPV genotype and the frequency of TILs or TCRs with HPV oncoprotein-T-cell cross-reactivity. Cross-reactivity was assessed by enzyme-linked immunospot assays and interferon-γ production assays. Results: The median (range) age of 65 referred patients was 44 (24-64) years. Ethnicity was recorded for 39 of 65 patients; 35 (89.7%) were white, 3 (7.7%) were Asian, and 1 (2.6%) was American Indian/Alaskan Native. Histologic tumor subtype was recorded for 41 of 65 patients; 25 (61.0%) were squamous cell carcinomas, 12 (29.3%) were adenocarcinomas, 2 (4.9%) were adenosquamous cell carcinomas, and 2 (4.9%) were neuroendocrine tumors. Thirty-nine of 65 patients (60.0%) had HPV-16-positive tumors and 21 patients (32.3%) had HPV-18-positive tumors. In the analysis of cross-reactivity, 1 of 16 oncoprotein-reactive archived TILs (9 from cervical cancers and 7 from other cancers) displayed HPV-16/HPV-18 cross-reactivity. None of the 10 oncoprotein-reactive TCRs displayed HPV-16/HPV-18 cross-reactivity. Conclusions and Relevance: Cervical cancers that tested positive for HPV-18 were common in this study and may be common in other US clinical trial populations. Results showed that HPV-16/HPV-18 intergenotype T-cell cross-reactivity of T cells from HPV-16-positive and HPV-18-positive cancers was uncommon. These findings support clinical trial designs in which the HPV type targeted by a therapeutic vaccine is matched with the HPV type of a cancer and suggest a change is necessary in the design of active clinical trials.

Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer.

Immunotherapy has clinical activity in certain virally associated cancers. However, the tumor antigens targeted in successful treatments remain poorly defined. We used a personalized immunogenomic approach to elucidate the global landscape of antitumor T cell responses in complete regression of human papillomavirus-associated metastatic cervical cancer after tumor-infiltrating adoptive T cell therapy. Remarkably, immunodominant T cell reactivities were directed against mutated neoantigens or a cancer germline antigen, rather than canonical viral antigens. T cells targeting viral tumor antigens did not display preferential in vivo expansion. Both viral and nonviral tumor antigen-specific T cells resided predominantly in the programmed cell death 1 (PD-1)-expressing T cell compartment, which suggests that PD-1 blockade may unleash diverse antitumor T cell reactivities. These findings suggest a new paradigm of targeting nonviral antigens in immunotherapy of virally associated cancers.

Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP.