The use of nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells

HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients\u27 serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious virions from infected samples

Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis

Objectives: 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. Method: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation. Results: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10-6). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e-15). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein. 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

Improved Detection of Herpesviruses from Diluted Vitreous Specimens Using Hydrogel Particles

Infectious uveitis is a sight-threatening infection commonly caused by herpesviruses. Vitreous humor is often collected for molecular confirmation of the causative agent during vitrectomy and mixed in large volumes of buffered saline, diluting the pathogen load. Here, we explore affinity-capture hydrogel particles (Nanotrap&reg;) to concentrate low abundant herpesviruses from diluted vitreous. Simulated samples were prepared using porcine vitreous spiked with HSV-1, HSV-2, VZV and CMV at 105 copies/mL. Pure undiluted samples were used to test capturing capability of three custom Nanotrap particles (red, white and blue) in a vitreous matrix. We found that all particles demonstrated affinity to the herpesviruses, with the Red Particles having both good capture capability and ease of handling for all herpesviruses. To mimic diluted vitrectomy specimens, simulated-infected vitreous were then serially diluted in 7 mL TE buffer. Diluted samples were subjected to an enrichment protocol using the Nanotrap Red particles. Sensitivity of pathogen detection by qPCR in diluted vitreous increased anywhere between 2.3 to 26.5 times compared to non-enriched specimens. This resulted in a 10-fold increase in the limit of detection for HSV-1, HSV-2 and VZV. These data demonstrated that Nanotrap particles can capture and concentrate HSV-1, HSV-2, VZV and CMV in a vitreous matrix

The use of Nanotrap particles in the enhanced detection of Rift Valley fever virus nucleoprotein.

Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics.After screening multiple Nanotrap particle architectures, we found that one particle, NT45, was optimal for RVFV NP capture, as demonstrated by western blotting. NT45 significantly enhanced detection of the NP at levels undetectable without the technology. Importantly, we demonstrated that Nanotrap particles are capable of concentrating NP in a number of matrices, including infected cell lysates, viral supernatants, and animal sera. Specifically, NT45 enhanced detection of NP at various viral titers, multiplicity of infections, and time points. Our most dramatic results were observed in spiked serum samples, where high abundance serum proteins hindered detection of NP without Nanotrap particles. Nanotrap particles allowed for sample cleanup and subsequent detection of RVFV NP. Finally, we demonstrated that incubation of our samples with Nanotrap particles protects the NP from degradation over extended periods of time (up to 120 hours) and at elevated temperatures (at 37ºC).This study demonstrates that Nanotrap particles are capable of drastically lowering the limit of detection for RVFV NP by capturing, concentrating, and preserving RVFV NP in clinically relevant matrices. These studies can be extended to a wide range of pathogens and their analytes of diagnostic interest

Nanotrap particles can capture RVFV NP.

<p>A) Recombinant histidine-tagged NP (His-NP) at a starting concentration of 0.4 mg/ml and a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at ambient temperature. After 30 minutes, the (+)NT samples were centrifuged. Both bound (P) and 10uL of unbound (S) material was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then analyzed for presence of NP protein by western blot using antibodies directed against the histidine tag. No Nanotrap particle samples (-NT) at a 10 μl volume of 0.4 mg/ml His-NP were processed in parallel. B) Purified NP (obtained from BEI Resources) at 2 μg in a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at an ambient temperature. A control—NT sample (10 μl volume) was processed in parallel. Samples were processed as describe in panel A. After electrophoresis, NP was visualized by Commassie blue staining. C) His-NP (obtained from Immune Technology) at 1 μg/ml and a volume of one milliliter was incubated with 100 μl of NT45, NT53, and NT69. A control—NT sample (10 μl volume) was processed in parallel. The control sample is 100 μg/ml NP (volume of 10 μl). The samples were processed as in panel A and analyzed by western blotting for NP by using antibodies against NP. D) Viral supernatants at 1E+06 pfu/ml and a volume of 1 ml were incubated with 100 μl NT45, NT46, NT53, NT55, and NT69 for 30 minutes at an ambient temperature. Control—NT samples (10 μl volumes) at 1E+07pfu/ml and 1E+06 pfu/ml were processed in parallel. The samples were processed as in panel A and analyzed by western blot for NP.</p

Non-virion associated NP can be detected in viral supernatants.

<p>Vivaspin 20 centrifugal concentrators with a 300,000 Da MWCO were used to filter viral supernatants harvested from Vero cells infected at an MOI 1 with MP12. Five milliliters of viral supernatant was added to the concentrators and centrifuged at 1400 rpm for 7 minutes until approximately 500 μl remained on the top portion of the concentrator. A) The top (T) and bottom (B) fractions, as well as control sample (C) containing the original sample before processing were analyzed by western blot using antibodies against NP (EC22 antibody). C, T, and B lysates were undiluted (neat). Cytoplasmic extract (CE) control is 7.7 μg/ml of RVFV infected Vero cell lysates. B) Plaque assays were performed with both fractions and the control sample. C) Densitometry analysis was performed to determine the NP band densities in the T and B fractions. The percent of total NP was determined by dividing the top or bottom portion's band density by the total NP band density (top and bottom portions added together) and multiplying by 100. D) NP band density per pfu values were calculated by dividing the NP band densities by the total pfu values.</p

Nanotrap particles can capture and enrich NP from virally infected cells.

<p>A) One ml of cytoplasmic extract (CE) at 2.6 μg/ml obtained from RVFV infected Vero lysates were incubated with 100 μl of NT45, NT53, and NT69. No Nanotrap particle sample (-NT) was processed in parallel. After 30 minutes, the (+)NT samples were centrifuged. The unbound material (S) from the spin was saved and 10 μl was processed in parallel. The bound material (P) was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then loaded onto a NuPage 4–12% Bis-Tris gel. Samples were subsequently analyzed by western blot for NP. B) One ml of CE was serially diluted in 50mM Tris-HCl from 15 μg/ml to 0.75 μg/ml and incubated with 100 μl of NT45 for 30 minutes. No Nanotrap particle samples (-NT) were processed in parallel. The control sample is CE at 770 μg/ml (10 μl volume). The samples were processed as in panel A.</p

Schematic of antigen capture with Nanotrap particles.

<p>Nanotrap particles are incubated with samples for 30 minutes at room temperature, centrifuged, and unbound material is removed. The pellet is resuspended in lysis buffer and boiled for 10 minutes. The sample is then centrifuged and unbound supernatant is loaded onto an SDS PAGE gel.</p

Nanotrap particles can protect NP from degradation at increased temperatures and times.

<p>A and B) Cytoplasmic extracts from RVFV infected cells were diluted in 100% sheep serum for a final concentration of 7.7 μg/ml. C and D) Viral supernatant was diluted in 100% goat serum for a final titer concentration of 1E+06 pfu/ml. One milliliter of sample was incubated with NT45 for 24 to 120 hours at either ambient temperature (A and C) or at 37^°C (B and D). No Nanotrap particle (-NT) and control cytoplasmic extract (CE) samples at 7.7μg/ml or 0.77 μg/ml (B only) at 10 μl volumes were processed in parallel. Viral supernatants without Nanotrap particles containing samples were analyzed on a separate gel (E). After the incubation time, the (+)NT samples were centrifuged and washed once with 0.25M sodium thiocyanate and twice with diH₂O. Presence of NP was analyzed by western blot using antibodies against NP.</p

Use of Nanotrap particles for the capture and enrichment of Zika, chikungunya and dengue viruses in urine.

Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses