POSTER Presented at American Society for Exosomes and Microvesicles (ASEMV) 2018 This work demonstrates that Nanotrap® particles can be used to enrich exosomes from tissue culture supernatant and cerebrospinal fluid (CSF). Using tissue culture supernatants, Nanotrap® particle enrichment of exosomes was confirmed by Western Blot, CD81 ELISA, acetylcholinesterase (AchE) activity, and flow cytometry. Nanotrap® particle enrichment of exosomes from CSF and PBMCs were confirmed by CD81 ELISA and Western Blot. Finally, using tissue culture supernatants and a CD81 ELISA, it was demonstrated that Nanotrap® particles can be used to enrich exosomes to equal or greater concentrations than exosomes isolated by ultra centrifugation from 10 times the volume of the same starting material. Access full poster here

POSTER - Abstract: HIV-1 is the causative agent of AIDS, infecting nearly 37 million people worldwide. Currently, no cure exists, mainly due to HIV-1’s ability to enact latency. Our previous work has shown that exosomes, small extracellular vesicles (EVs), from uninfected cells can activate HIV-1 in latent cells, leading to increased mostly short and some long HIV-1 RNA transcripts. This is consistent with the notion that none of the FDAapproved antiretroviral drugs used today in the clinic are transcription inhibitors. Furthermore, these HIV-1 transcripts can be packaged into EVs and released from the infected cell. In this study, we investigated the specific mechanism behind the activation of latent HIV-1 by EVs. We observed that the EV-associated kinase c-Src is able to activate latent HIV-1 in infected cells via the PI3K/AKT/mTOR pathway and SRC1/p300-driven chromatin remodeling. We discovered that upon inhibiting each of the proteins involved in the PI3K/AKT/mTOR pathway, HIV-1 transcription, as well as levels of HIV-1 Gag p24 in the cell supernatant, was decreased. Collectively, our data suggests that the EV activation of latent HIV-1 is initially started by EV-associated c-Src being delivered into a recipient cell, where it is able to activate the PI3K/AKT/mTOR pathway, eventually leading to the activation and translocation of SRC-1 to the nucleus, promoting a pro-transcription state. Access full poster here

Clinical and Translational Medicine, 2018 Viral antigens detectable in CSF exosomes from patients with retrovirus associated neurologic disease: functional role of exosomes Background: HTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences. Results: Exosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12), (Fisher exact test, p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis. Conclusion: Cumulatively, these results show that there are HTLV-1 proteins present in exosomes found in virus-free CSF. HAM/TSP PBMCs, particularly CD4+ CD25+ T cells, can excrete these exosomes containing HTLV-1 Tax and may be a source of the exosomes found in patient CSF. Importantly, these exosomes are capable of sensitizing an HTLV-1 specific immune response, suggesting that they may play a role in the immunopathology observed in HAM/TSP. Given the infiltration of HTLV-1 Tax-specific CTLs into the CNS of HAM/TSP patients, it is likely that exosomes may also contribute to the continuous activation and inflammation observed in HAM/TSP, and may suggest future targeted therapies in this disorder. https://clintransmed.springeropen.com/articles/10.1186/s40169-018-0204-7

Scientific Reports, 2018 Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells Introduction: To date, the most effective treatment of HIV-1 is a combination antiretroviral therapy (cART), which reduces viral replication and reverses pathology. We investigated the effect of cART (RT and protease inhibitors) on the content of extracellular vesicles (EVs) released from HIV-1-infected cells. We have previously shown that EVs contain non-coding HIV-1 RNA, which can elicit responses in recipient cells. In this manuscript, we show that TAR RNA levels demonstrate little change with the addition of cART treatment in cell lines, primary macrophages, and patient biofluids. We determined possible mechanisms involved in the selective packaging of HIV-1 RNA into EVs, specifically an increase in EV associated hnRNP A2/B1. More recent experiments have shown that several other FDA-approved drugs have the ability to alter the content of exosomes released from HIV-1-infected cells. These findings on cART-altered EV content can also be applied to general viral inhibitors (interferons) which are used to treat other chronic infections. Additionally, we describe unique mechanisms of ESCRT pathway manipulation by antivirals, specifically the targeting of VPS4. Collectively, these data imply that, despite antiretroviral therapy, EVs containing viral products are continually released and may cause neurocognitive and immunological dysfunction.

Download the Poster Use of Nanotrap® Particles for capture and enrichment of febrile-illness-causing pathogens, including Zika, Chikungunya, Dengue, and Influenza viruses Presented at ASM CVS 2018 We assessed the utility of Nanotrap Microbiome Particles for capture and enrichment of febrile-illness causing pathogens, including Zika (ZIKV), chikungunya (CHIKV), dengue (DENV), and influenza (FLU) viruses. Through the use of qRT-PCR methods, we demonstrated that Nanotrap Particle type CN1030 provides at least 8-fold enrichment of ZIKV, CHIKV, and DENV from urine biospecimens. We demonstrated detection of ZIKV, CHIKV, and DENV at viral concentrations as low as 0.1 PFU/mL.

Expert Review of Proteomics, 2018 Affinity enrichment for mass spectrometry: improving the yield of low abundance biomarkers ​ Introduction: Mass spectrometry (MS) is the premier tool for discovering novel disease-associated protein biomarkers. Unfortunately, when applied to complex body fluid samples, MS has poor sensitivity for the detection of low abundance biomarkers (≪10 ng/mL), derived directly from the diseased tissue cells or pathogens. Areas covered: Herein we discuss the strengths and drawbacks of technologies used to concentrate low abundance analytes in body fluids, with the aim to improve the effective sensitivity for MS discovery. Solvent removal by dry-down or dialysis, and immune-depletion of high abundance serum or plasma proteins, is shown to have disadvantages compared to positive selection of the candidate biomarkers by affinity enrichment. A theoretical analysis of affinity enrichment reveals that the yield for low abundance biomarkers is a direct function of the binding affinity (Association/Dissociation rates) used for biomarker capture. In addition, a high affinity capture pre processing step can effectively dissociate the candidate biomarker from partitioning with high abundance proteins such as albumin. Expert commentary: Properly designed high affinity capture materials can enrich the yield of low abundance (0.1–10 picograms/mL) candidate biomarkers for MS detection. Affinity capture and concentration, as an upfront step in sample preparation for MS, combined with MS advances in software and hardware that improve the resolution of the chromatographic separation can yield a transformative new class of low abundance biomarkers predicting disease risk or disease latency.

Nanomedicine, 2018 Toward detection of toxoplasmosis from urine in mice using hydro-gel nanoparticles concentration and parallel reaction monitoring mass spectrometry ​ Diagnosis of clinical toxoplasmosis remains a challenge, thus limiting the availability of human clinical samples. Though murine models are an approximation of human response, their definitive infection status and tissue availability make them critical to the diagnostic development process. Hydrogel mesh nanoparticles were used to concentrate antigen to detectable levels for mass spectrometry. Seven Toxoplasma gondii isolates were used to develop a panel of potential peptide sequences for detection by parallel reaction monitoring (PRM) mass spectrometry. Nanoparticles were incubated with decreasing concentrations of tachyzoite lysate to explore the limits of detection of PRM. Mice whose toxoplasmosis infection status was confirmed by quantitative real-time PCR had urine tested by PRM after hydrogel mesh concentration for known T. gondii peptides. Peptides from GRA1, GRA12, ROP4, ROP5, SAG1, and SAG2A proteins were detected by PRM after nanoparticle concentration of urine, confirming detection of T. gondii antigen in the urine of an infected mouse.

Colloids and Surfaces B: Biointerfaces, 2018 Structurally stable N-t-butylacrylamide hydrogel particles for the capture of peptides Hydrogel particles have proven to be powerful tools for the capture and concentration of low abundance, low molecular weight peptides and proteins from complex biofluids, such as plasma. The primary means of recovering and washing the particles following harvesting is through centrifugation, which can be a very time-consuming process depending on harvest conditions. To improve the process of particle recovery, washing, and elution we have developed new particle formulations: incorporating N-t-butylacrylamide (tBA) in the polymer backbone with monomers bearing more acidic functional groups and higher degrees of cross-linking. These particle formulations produce a stable architecture that does not significantly respond to changes in environmental conditions, such as pH and temperature. These two new formulations impart structural stability to the particle, control swelling, and improve pelleting through centrifugation, even at high pH values. These structurally stable microparticles yield improved particle recovery while maintaining the peptide capture properties of the particle.

Science Translational Medicine, 2017 Urine lipoarabinomannan glycan in HIV-negative patients with pulmonary tuberculosis correlates with disease severity Lipoarabinomannan (LAM) is a component of the cell wall shed by Mycobacterium tuberculosis, the bacteria responsible for tuberculosis, an infection mainly affecting the lungs. LAM can be detected in urine samples from patients coinfected with HIV, but current LAM detection methods have failed for HIV-negative patients. Using hydrogel Nanotrap® particles ("Nanocages") and a chemical bait with high affinity for LAM, Paris et al. showed that patients negative for HIV with active tuberculosis infections had detectably higher concentrations of LAM in their urine than patients without active tuberculosis infections. Nanocages could also be used to detect cytokines and other antigens present in low concentrations in urine, demonstrating the versatility of the technology as a method to detect and monitor infections.

Frontiers in Molecular Biosciences, 2017: Immune-modulating Activity of Hydrogel Microparticles Contributes to the Host Defense in a Murine Model of Cutaneous Anthrax In prior study, it was shown that the open-mesh (0.7 μ) polyacrylamide microparticles (MPs) with internally-coupled Cibacron affinity dye demonstrate protective effect in mice challenged into footpads with high doses (200 LD50) of anthrax (Sterne) spores. A single injection of MPs before spore challenge reduces inflammatory response, delays onset of mortality and promotes survival. In this study, we show that the effect of MPs was substantially increased at the lower spore dose (7 LD50). The inflammation of footpads was reduced to the background level, and 60% of animals survived for 16 days while all untreated infected animals died within 6 days with strong inflammation. The effects of MPs were promoted when the MPs were loaded with a combination of neutrophil-attracting chemokines IL-8 and MIP-1a which delayed the onset of mortality in comparison with untreated mice for additional 8 days. The MPs were not inherently cytotoxic against the bacteria or cultured murine Raw 264.7 cells, but stimulated these cells to release G-CSF, MCP-1, MIP-1a, and TNF-a. Consistent with this finding the injection of MPs induced neutrophil influx into footpads, stimulated production of TNF-a associated with migration of pERK1/2-positive cells with the Langerhans phenotype from epidermis to regional lymph nodes. Our data support the mechanism of protection in which the immune defense induced by MPs along with the exogenous chemokines counterbalances the suppressive effect caused by anthrax infection. In conclusion, our findings show for the first time that the poly(N-isopropylacrylamide) open-mesh MPs containing copolymerized allylamine or covalently-bound CB dye behave as potent immune stimulators. The dye-coupled MPs were capable of altering the course of infectious disease, and their potency was enhanced by loaded CKs. Detailed examination of the specific roles the immune cell types and their subpopulations, including the DCs, play in the effect of MPs is forthcoming. It will be intriguing to evaluate the effect of MPs with different coupled (surface or external) chemistries against pathogenic bacteria other than B.a. used under different modes of MP administration. See full publication here

Journal of Biological Chemistry, 2017 Exosomes from uninfected cells activate transcription of latent HIV-1 HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1–infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR-gag, can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo, especially in the presence of combination antiretroviral therapy.

​ POSTER LabCorp, SM Clinical Virology Symposium,  May 8, 2017 Purification and Conservation of Cytomegalovirus (CMV) Antibody in Oral Fluid Using NanoTrap® Particle Technology ​ Routine serological testing for antibodies to detect current or past exposure to infectious agents requires the collection of a blood sample by venipuncture, limiting sample collection to conventional healthcare settings. Using non-invasive samples for such testing is highly desirable, since it enables expansion of test access to non-conventional settings, such as health fairs and donor drives. Oral fluid, specifically mucosal transudate, contains measurable concentrations of immunoglobulins and has been used successfully as an alternative to serum for a limited number of applications. The content of oral fluid collections is highly variable, however, and the presence of interferents (e.g. mucous, saliva, microbes) can impact both the specificity and sensitivity of immunoassays optimized for serum or plasma samples. This has typically necessitated the development of assays that are specifically designed for using oral fluid as the test matrix. The availability of a pre-analytical technology that eliminates background interferents whilst simultaneously enriching the sample for the target analyte could potentially allow oral fluid to be widely utilized as a sample type for enzyme immunoassays (EIA) developed originally for blood-based testing. In this study, we demonstrate the ability of such a technology, namely the NanoTrap® system (Ceres Nanosciences; Manassas, VA), to concentrate and purify Cytomegalovirus (CMV) IgG from oral fluid collections enabling successful identification of seropositive individuals without the need for blood collection.