Journal of Proteome Research, 2017 Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043.

POSTER Background: Ebola virus (EBOV) mainly targets cells of the myeloid lineage; however, extensive death of T-cells takes place in lethal infections. We have previously shown that EBOV VP40 in extracellular vesicle (EVs) causes recipient cell death in immune cells. Methods: Using VP40-producing cell lines, we analyzed donor cell cycle, EV biogenesis, and recipient immune cell viability. EV contents were characterized by mass spectrometry, cytokine array, and Western blot. BSL-4 facilities were utilized for wild-type Ebola virus infection studies. Results: VP40 EVs induced cell death by apoptosis in recipient T-cells and monocytes. VP40- producing cells had accelerated cell cycling, which was tied to EV biogenesis, resulting in fewer but larger EVs. VP40 EVs were enriched in several cytokines, including IL-15, TGF-β1, and IFN-γ. Finally, EBOV-infected cell and animal EVs contained VP40, NP, and GP. Conclusions: EBOV VP40 resulted in dysregulated cell cycle and EV biogenesis in donor cells. Packaging of cytokines and EBOV proteins into EVs from infected cells may be responsible for the decimation of immune cells during EBOV pathogenesis. Access full poster here

Frontiers in Microbiology 2016 Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80-90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, we examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. Additionally, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the deregulation and eventual destruction of the T-cell and myeloid arms of the immune system (bystander lymphocyte apoptosis), allowing the virus to replicate to high titers in the immunocompromised host. Moreover, our results suggest that the use of drugs such as Oxytetracycline to modulate the levels of exosomes exiting EBOV-infected cells may be able to prevent the devastation of the adaptive immune system and allow for an improved rate of survival.

Virulance, 2016 Enhanced detection of respiratory pathogens with Nanotrap® particles The Influenza virus is a leading cause of respiratory disease in the United States each year. While the virus normally causes mild to moderate disease, hospitalization and death can occur in many cases. There are several methodologies that are used for detection; however problems such as decreased sensitivity and high rates of false-negative results may arise. There is a crucial need for an effective sample preparation technology that concentrates viruses at low abundance while excluding resident analytes that may interfere with detection. Nanotrap® particles are hydrogel particles that are coupled to chemical dye affinity baits that bind a broad range of proteins and virions. Within minutes (<30 minutes), Nanotrap® particles concentrate low abundant proteins and viruses from clinically complex matrices. Nanotrap particles with reactive red baits concentrated numerous respiratory viruses including various strains and subtypes of Influenza virus, Coronavirus, and Respiratory Syncytial Virus from saliva, nasal fluid swab specimens, and nasal aspirates. Detection was enhanced more than 10-fold when coupled to plaque assays and qRT-PCR. Importantly, Nanotrap® particles can efficiently capture and concentrate multiple viral pathogens during a coinfection scenario. These results collectively demonstrate that Nanotrap® particles are an important tool that can easily be integrated into various detection methodologies.

PLOS One, 2016 Chemokine-Releasing Microparticles Improve Bacterial Clearance and Survival of Anthrax Spore-Challenged Mice In this study the hydrogel microparticles (MPs) were used to enhance migration of neutrophils in order to improve outcome of anthrax infection in a mouse model. Two MP formulations were tested. In the first one the polyacrylamide gel MPs were chemically coupled with Cibacron Blue (CB) affinity bait. In the second one the bait molecules within the MPs were additionally loaded with neutrophil-attracting chemokines (CKs), human CXCL8 and mouse CCL3. A non-covalent interaction of the bait with the CKs provided their gradual release after administration of the MPs to the host. Mice were challenged into footpads with Bacillus anthracis Sterne spores and given a dose of MPs a few hours before and/or after the spores. Pre-treatment with a single dose of CK-releasing MPs without any additional intervention was able to induce influx of neutrophils to the site of spore inoculation and regional lymph nodes correlating with reduced bacterial burden and decreased inflammatory response in footpads. On average, in two independent experiments, up to 53% of mice survived over 13 days. All control spore-challenged but MP-untreated mice died. The CB-coupled particles were also found to improve survival likely due to the capacity to stimulate release of endogenous CKs, but were less potent at decreasing the inflammatory host response than the CK-releasing MPs. The CK post-treatment did not improve survival compared to the untreated mice which died within 4 to 6 days with a strong inflammation of footpads, indicating quick dissemination of spores though the lymphatics after challenge. This is the first report on the enhanced innate host resistance to anthrax in response to CKs delivered and/or endogenously induced by the MPs.

POSTER Sample preparation and stabilization are critically important to downstream diagnostic assay sensitivity, as they directly impact the abundance and detection of analytes. Moreover, the Warfighter requires solutions for diagnostic sample processing that are simple, fast, and robust when operating in austere environments. Nanotraps® are hydrogel nanoparticles functionalized with chemical affinity baits that can capture low abundance proteins, peptides, metabolites, nucleic acids, small molecules, and whole virions, while also reducing high abundance interfering substances. Previously published studies have shown that Nanotraps can capture both virions and proteins from influenza virus, Rift Valley fever virus, HIV, and Venezuelan equine encephalitis virus (VEEV). Here, we applied Nanotrap® technology to Ebola virus (EBOV) diagnostic applications and found that Nanotrap® sample processing increased assay sensitivity for EBOV-specific nucleic acid tests and protein immunoassays in spiked human clinical matrices, and in matrices collected from nonhuman primate models of EBOV infection. Access full poster here

Frontiers in Microbiology, Feb 2016 Presence of Viral RNA and Proteins in Exosomes from Cellular Clones Resistant to Rift Valley Fever Virus Infection Rift Valley Fever Virus (RVFV) is a RNA virus that belongs to the genus Phlebovirus, family Bunyaviridae. It infects humans and livestock and causes Rift Valley fever. RVFV is considered an agricultural pathogen by the USDA, as it can cause up to 100% abortion in cattle and extensive death of newborns. In addition, it is designated as Category A pathogen by the CDC and the NIAID. In some human cases of RVFV infection, the virus causes fever, ocular damage, liver damage, hemorrhagic fever, and death. There are currently limited options for vaccine candidates, which include the MP-12 and clone 13 versions of RVFV. Viral infections often deregulate multiple cellular pathways that contribute to replication and host pathology. We have previously shown that latent human immunodeficiency virus-1 (HIV-1) and human T-cell lymphotropic virus-1 (HTLV-1) infected cells secrete exosomes that contain short viral RNAs, limited number of genomic RNAs, and viral proteins. These exosomes largely target neighboring cells and activate the NF-κB pathway, leading to cell proliferation, and overall better viral replication. In this manuscript, we studied the effects of exosome formation from RVFV infected cells and their function on recipient cells. We initially infected cells, isolated resistant clones, and further purified using dilution cloning. We then characterized these cells as resistant to new RVFV infection, but sensitive to other viral infections, including Venezuelan Equine Encephalitis Virus (VEEV). These clones contained normal markers (i.e., CD63) for exosomes and were able to activate the TLR pathway in recipient reporter cells. Interestingly, the exosome rich preparations, much like their host cell, contained viral RNA (L, M, and S genome). The RNAs were detected using qRT-PCR in both parental and exosomal preparations as well as in CD63 immunoprecipitates. Viral proteins such as N and a modified form of NSs were present in some of these exosomes. Finally, treatment of recipient cells (T-cells and monocytic cells) showed drastic rate of apoptosis through PARP cleavage and caspase 3 activation from some but not all exosome enriched preparations. Collectively, these data suggest that exosomes from RVFV infected cells alter the dynamics of the immune cells and may contribute to pathology of the viral infection.

Journal of Biological Chemistry, 2016 Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA ​ HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/μl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-β, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.

Journal of Translational Medicine, 2015 Application of Nanotrap® technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap® particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB. Conclusions: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.

Frontiers in microbiology, 2015 Extracellular vesicles from infected cells: potential for direct pathogenesis Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogen-associated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFκB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts.

POSTER Neurology, 2015 Detection of Human T-cell Lymphotropic Virus Type I proteins in exosomes from HAM/TSP patient CSF by novel Nanotrap® technology OBJECTIVE: To assess the presence of viral proteins in the CSF of patients with HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) BACKGROUND: There has been increasing evidence for the role of microvesicles (MV) in CNS inflammation and viral disease. HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disease that affects a subset of virus-infected infected individuals. This disorder is immunopathologically mediated since virus -specific immune cells can be found in both the CSF and in the CNS of patients although HTLV-I virus has been difficult to isolate in CNS resident cells. The possibility of transfer of viral proteins via MV from viral reservoirs to uninfected cells in the absence of virus is an intriguing mechanism by which this can occur. Recently, HTLV-1 tax proteins have been shown in exosomes (EX) from HTLV-1 infected cell lines (Jaworksi et al 2014). Therefore, we examined if similar MV were present in the CNS of HAM/TSP patients. DESIGN/METHODS: HTLV-1 infected and uninfected cell line supernatants were used for isolation of EX and MV by a novel Nanotrap® (NT) technology. These NTs are hydrogel particles consisting of high affinity aromatic baits surrounded by a sieving shell. HTLV-I viral proteins were assessed by Western blot analysis. HTLV-I virions were detected by RT-qPCR. In addition, EXs were isolated using this EX-NT from CSF of HAM/TSP patients and MS patients as controls. RESULTS: Exosomes were successfully isolated specifically from HTLV-1 infected cell lines that contained HTLV-I tax protein but lacked HTLV-I virions. Moreover, CSF from HAM/TSP patients and not from MS, also demonstrated exosomes that were HTLV-I tax Western blot positive. CONCLUSIONS: These results suggest the possibility that HTLV-I protein present in virus-free CSF can be a potential source of antigen in an inflammatory neuropathological disease.

PLOS Neglected Tropical Diseases, 2013 Use of a Novel Chagas Urine Nanoparticle Test for Diagnosis of Congenital Chagas Disease Detection of congenital T. cruzi transmission is considered one of the pillars of control programs of Chagas disease. Congenital transmission accounts for 25% of new infections with an estimated 15,000 infected infants per year. Current programs to detect congenital Chagas disease in Latin America utilize microscopy early in life and serology after 6 months. These programs suffer from low sensitivity by microscopy and high loss to follow-up later in infancy. We developed a Chagas urine nanoparticle test (Chunap) to concentrate, preserve and detect T. cruzi antigens in urine for early, non-invasive diagnosis of congenital Chagas disease.