A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.

Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays, and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microscale benefits of rapid magnetic separation. This system comprises magnetic nanoparticles and polymer-antibody (Ab) conjugates that transition from hydrophilic nanoscale reagents to microscale aggregates in response to temperature stimuli. The binary reagent system was benchmarked against Ab-labeled Dynabeads in terms of biomarker isolation kinetics, assay speed, and reagent needs. Surface plasmon resonance (SPR) measurements showed that polymer conjugation did not significantly alter the Ab's binding affinity or kinetics. ELISA analysis showed that the unconjugated Ab, polymer-Ab conjugates, and Ab-labeled Dynabeads exhibited similar equilibrium dissociation constants (Kd), ∼2 nM. However, the binary reagent system isolated HIV p24 antigen from spiked serum specimens (150 pg/mL) much more quickly than Dynabeads, which resulted in shorter binding times by tens of minutes, or about 30-50% shorter overall assay times. The binary reagent system showed improved performance because the Ab molecules were not conjugated to large, solid microparticle surfaces. This stimuli-responsive binary reagent system illustrates the potential advantages of nanoscale reagents in molecule and cell isolations for both research and clinical applications.

Endosomal trafficking of nanoformulated antiretroviral therapy facilitates drug particle carriage and HIV clearance.

UNLABELLED: Limitations of antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance, and failure to penetrate viral reservoirs. Recent developments in nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at or adjacent to the sites of the viral replication cycle. Poloxamer 407-coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were ∼1,000 times higher in cells than those that could be achieved by the native drug. ATV particles in late and recycling endosome compartments were seen following pulldown by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of the compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1 p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000-fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The colocalization of nanoATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long-acting nanoART can serve as a drug carrier in both cells and subcellular compartments and, as such, can facilitate viral clearance. IMPORTANCE: The need for long-acting ART is significant and highlighted by limitations in drug access, toxicity, adherence, and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells, and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly, and increasing ART concentrations in such sites enhances viral clearance. The current work uncovers a new mechanism by which nanoART can enhance viral clearance over native drug formulations.

Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system.

BACKGROUND: The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. RESULTS: A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose-response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. CONCLUSIONS: The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system.

Controlled copper in situ growth-amplified lateral flow sensors for sensitive, reliable, and field-deployable infectious disease diagnostics.

A polyethyleneimine (PEI)-assisted copper in-situ growth (CISG) strategy was proposed as a controlled signal amplification strategy to enhance the sensitivity of gold nanoparticle-based lateral flow sensors (AuNP-LFS). The controlled signal amplification is achieved by introducing PEI as a structure-directing agent to regulate the thermodynamics of anisotropic Cu nanoshell growth on the AuNP surface, thus controlling shape and size of the resultant AuNP@Cu core-shell nanostructures and confining free reduction and self-nucleation of Cu2+ for improved reproducibility and decreased false positives. The PEI-CISG-enhanced AuNP-LFS showed ultrahigh sensitivities with the detection limits of 50 fg mL-1 for HIV-1 capsid p24 antigen and 6 CFU mL-1 for Escherichia coli O157:H7. We further demonstrated its clinical diagnostic efficacy by configuring PEI-CISG into a commercial AuNP-LFS detection kit for SARS-CoV-2 antibody detection. Altogether, this work provides a reliable signal amplification platform to dramatically enhance the sensitivity of AuNP-LFS for rapid and accurate diagnostics of various infectious diseases.

A multi-walled carbon nanotubes based molecularly imprinted polymers electrochemical sensor for the sensitive determination of HIV-p24.

To develop a rapid, simple and sensitive method for the determination of human immunodeficiency virus p24 (HIV-p24), a novel molecularly imprinted polymers (MIPs) electrochemical sensor was constructed on the surface of a multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE) by surface polymerization using acrylamide (AAM) as functional monomer, N,N'-methylenebisacrylamide (MBA) as cross-linking agent and ammonium persulphate (APS) as initiator. Each modification step was carefully examined by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and scanning electron microscope (SEM). The proposed MIPs electrochemical biosensor exhibited specific recognition to HIV-p24 and displayed a broad linear detection range from 1.0×10-4 to 2ngcm-3 with a low detection limit of 0.083pgcm-3 (S/N=3). This performance is superior to most HIV-p24 sensors based on other methods. Meanwhile, this sensor possessed of good selectivity, repeatability, reproducibility, stability and was successfully applied for the determination of HIV-p24 in real human serum samples, giving satisfactory results. The accuracy and reliability of the sensor is further confirmed by enzyme-linked immunosorbent assay (ELISA).

Flow-through, viral co-infection assay for resource-limited settings.

Here we present a new and rapid immunofiltration assay for simultaneous detection of HIV p24 and hepatitis B virus antigens. The assay platform is composed of a 13 mm nitrocellulose filter spotted with capturing bioprobes and inserted in a Swinnex(®) syringe filter holder. Samples and reagents are flown through the nitrocellulose filter by manual pressure on the syringe. A colorimetric detection allows for naked eye results interpretation. The assay provides sensitivity in the picomolar range in just 5 min, even using low volumes of sample in complex matrix. Probe deposition by spotting allows for flexible combinations of different capturing agents and multiple diagnoses; furthermore, the very simple and inexpensive set-up makes the syringe-based immunoassay on paper microarray a suitable diagnostic system for resource-limited settings.

Antiviral efficacy, intracellular uptake and pharmacokinetics of free and liposome-encapsulated 2',3'-dideoxyinosine.

OBJECTIVE: To evaluate the effect of liposome encapsulation on the in vitro antiviral efficacy, intracellular uptake and in vivo pharmacokinetics of 2',3'-dideoxyinosine (ddl). METHODS: The accumulation of free and liposome-encapsulated ddl was determined in murine monocyte-macrophage RAW 264.7 cells and human premonocytoid U937 cells. The antiviral efficacy was evaluated in U937 cells infected with HIVIIIB. Tissue distribution and pharmacokinetics of free and liposomal ddl were determined in female Sprague-Dawley rats following the administration of a single intravenous bolus dose (3 mg ddl/kg). RESULTS: The entrapment of ddl in liposomes results in a lower drug accumulation in both U937 and RAW 264.7 cells. A lower antiviral efficacy against HIVIIIB replication in U937 cells was observed on encapsulation of ddl in liposomes. Improved pharmacokinetics were observed on entrapment of ddl in liposomes. Higher drug levels were found in plasma for the liposomal formulation. The systemic clearance of the liposomal drug was 120 times lower than that of free drug. Liposome encapsulation of ddl greatly enhanced the drug accumulation in organs of the reticuloendothelial system. CONCLUSION: The encapsulation of ddl in liposomes modified the tissue distribution and plasma pharmacokinetics of the antiviral agent resulting in a marked improvement of drug biodisponibility. The antiviral efficacy of liposomal ddl was lower than that of free drug in HIVIIIB-infected U937 cells.

Effects of fixation and paraffin embedding on the immunohistological detection of cell-associated HIV-1 by different monoclonal antibodies.

This study evaluates a panel of monoclonal antibodies (MAbs) to HIV-1 antigens (DuPont anti-gp120, gp41, p24; Olympus anti-gp120/160, gp41, p24A, p24B, p55, p18A, p18B, reverse transcriptase) for their ability to detect the virus in tissues after exposure to various fixatives (100% acetone, 10% formaldehyde, 2.5% glutaraldehyde, 4% paraformaldehyde/1% glutaraldehyde, Bouin's fluid) and after paraffin embedding. Acetone, 10% formaldehyde, and Bouin's fluid all preserved a wide range of viral epitopes compared with other fixatives. The most robust MAbs were DuPont p24 and Olympus p55, which produced excellent staining regardless of the fixative used. Embedding in paraffin variability influenced the capacity of MAbs to detect HIV-1 epitopes on fixed cells. Certain antibodies (e.g., DuPont gp24, Olympus p24B) produced good staining, whereas other epitopes (e.g., DuPont gp120, formaldehyde) were destroyed. In some cases, paraffin embedding revealed antigenic sites that had been formerly masked (e.g., Olympus gp120 and p24A; formaldehyde and glutaraldehyde fixation). These results indicate that HIV-1 antigens can be detected by immunohistology on cells exposed to most common fixatives. Therefore, retrospective analysis of pathological material is possible, provided that the antibodies are matched to the fixative used to preserve the tissue.

Infectious immune complexes in HIV-1-infected patients.

Using polyethylene glycol (PEG) precipitation we have found that most HIV-1 seropositive patients have IgG containing-circulating immune complexes (CIC). In addition these CIC sometimes contain IgA, IgM, C3, and/or HIV p24 antigen. Previous work has demonstrated that patients who have plasma viremia, CD4 cell counts less than 170/mm3, or who are symptomatic are more apt to have HIV that is precipitable with PEG. In this study we report that the infectious HIV found in the plasma of patients with plasma viremia could only be found in the 2% PEG precipitates, i.e., PEG supernatants never contained infectious HIV, although they often contained noninfectious p24 antigen. These results suggested that at least some of the infectious HIV circulating in the plasma of infected patients is in the form of immune complexes. To support this idea we also demonstrated that infectious HIV could be precipitated with antiserum raised to either immunogloblins or complement components.

Human submandibular saliva specifically inhibits HIV type 1.

Studies from a number of laboratories have shown the presence of factor(s) in whole, parotid, and submandibular human saliva capable of inhibiting HIV-1 infectivity in vitro. Data from our laboratory suggested that the level of anti-HIV-1 activity is higher in submandibular than parotid or whole saliva. Previous results obtained with pooled submandibular saliva from seronegative individuals included a filtration step following saliva-virus interaction. In this article, we present data on the HIV-1 inhibitory activity of individual submandibular saliva samples collected from 15 donors. We show that although anti-HIV activity is quantitatively similar in most individuals (9 of 15), some (4 of 15) are much less active than others and some (2 of 15) lack inhibitory activity. We also show that for most individuals the level of anti-HIV inhibitor is similar with or without a filtration step. However, 2 of the 15 samples demonstrated activity only after filtration. The quantitative and qualitative anti-HIV activity of individual saliva samples appeared to reflect differences in the individual donors. We further show that the anti-HIV activity of submandibular saliva is demonstrated not only against laboratory strains of HIV-1 but is similarly active against three clinical HIV-1 isolates. In contrast, submandibular saliva had little effect on the infectivity of HIV-2 or SIV.

Evaluation of anti-AIDS drugs in conventional mice implanted with a permeable membrane device containing human T cells infected with HIV.

We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.

Necrotizing ulcerative stomatitis in human immunodeficiency virus-seropositive individuals: a review of the histopathologic, immunohistochemical, and virologic characteristics of 18 cases.

OBJECTIVE: The purpose of this retrospective study was to delineate the histopathologic, immunohistochemical, and virologic characteristics of 18 cases of necrotizing ulcerative stomatitis. STUDY DESIGN: Eighteen examples or oral ulcerations in human immunodeficiency virus-seropositive individuals were identified that displayed unique histopathologic features. Immunohistochemic staining for CD1a, CD3, CD23, CD68, HLA-DR, p24, cytomegalovirus, HSV-1, and HSV-2 was performed, along with in situ hybridization for Epstein-Barr virus RNA and special staining for bacteria and fungi. RESULTS: The lesions demonstrated ulceration, extensive necrosis, leukocytoclasia, histiocytic vasculitis with luminal fibrin clots, and a prominent infiltrate of large atypical cells with amphophilic cytoplasm, vesicular nuclei, and prominent nucleoli, interspersed with crescentic histiocytes, a histologic picture resembling extranodal Kikuchi's disease. Immunohistochemical findings suggested that the large atypical cells were histiocytes. Fifty-six percent (10/18) of the cases were immunoreactive for human immunodeficiency virus p24 within focal histiocytes, whereas Epstein-Barr virus RNA was identified in 1 (6%) of 17 cases. CONCLUSIONS: Necrotizing ulcerative stomatitis is an inflammatory disease characterized by specific, reproducible microscopic features. We postulate that the histopathologic resemblance of necrotizing ulcerative stomatitis to extranodal Kikuchi's disease reflects a similar immune response to differing pathogens.

Recovery of infectious HIV-1 from whole saliva.

Whole saliva and serum samples were collected from 75 HIV-infected homosexual or bisexual men. Thirty-eight percent of cultured sera were positive for cell-free, infectious virus while only 1 percent of the 218 cultured whole salivas contained cell-free, infectious virus. These data support previous studies suggesting unlikely potential transmissibility of HIV infection by saliva.

Layer-by-layer coated digitally encoded microcarriers for quantification of proteins in serum and plasma.

The "layer-by-layer" (LbL) technology has been widely investigated for the coating of flat substrates and capsules with polyelectrolytes. In this study, LbL polyelectrolyte coatings applied at the surface of digitally encoded microcarriers were evaluated for the quantitative, sensitive, and simultaneous detection of proteins in complex biological samples like serum, plasma, and blood. LbL coated microcarriers were therefore coupled to capture antibodies, which were used as capture agents for the detection of tumor necrosis factor (TNF-alpha), P24, and follicle stimulating hormone (FSH). It was found that the LbL coatings did not disassemble upon incubating the microcarriers in serum and plasma. Also, nonspecific binding of target analytes to the LbL coating was not observed. We showed that the LbL coated microcarriers can reproducibly detect TNF-alpha, P24, and FSH down to the picogram per milliliter level, not only in buffer but also in serum and plasma samples. Microcarrier-to-microcarrier intratube variations were less then 30%, and interassay variations less than 8% were observed. This paper also shows evidence that the LbL coated digitally encoded microcarriers are ideally suited for assaying proteins in "whole" blood in microfluidic chips, which are of high interest for "point-of-care" diagnostics.

Decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1.

The Gag-Pol polyprotein of the human immunodeficiency virus type 1 (HIV-1) is the precursor of the virus enzymatic activities and is produced via a programmed -1 translational frameshift. In this study, we altered the frameshift efficiency by introducing mutations within the slippery sequence and the frameshift stimulatory signal, the two elements that control the frameshift. These mutations decreased the frameshift efficiency to different degrees, ranging from approximately 0.3% to 70% of the wild-type efficiency. These values were mirrored by a reduced incorporation of Gag-Pol into virus-like particles, as assessed by a decrease in the reverse transcriptase activity associated to these particles. Analysis of Gag processing in infectious mutant virions revealed processing defects to various extents, with no clear correlation with frameshift decrease. Nevertheless, the observed frameshift reductions translated into equivalently reduced viral infectivity and replication kinetics. Our results show that even moderate variations in frameshift efficiency, as obtained with mutations in the frameshift stimulatory signal, reduce viral replication. Therapeutic targeting of this structure may therefore result in the attenuation of virus replication and in clinical benefit.

The anti-HIV activity of ADS-J1 targets the HIV-1 gp120.

Recent data suggest that heparin sulfates may bind to a CD4 induced epitope in the HIV-1 gp120 that constitutes the coreceptor binding site. We have studied the mechanism of action of ADS-J1, a non-peptidic compound selected by docking analysis to interact with gp41 and to interfere with the formation of N-36/C-34 complexes in sandwich ELISA experiments. We show that ADS-J1 blocked the binding of wild-type HIV-1 NL4-3 strain to MT-4 cells but not virus-cell binding of a polyanion-resistant virus. However, ADS-J1 blocked the replication of polyanion-resistant, T-20- and C34-resistant HIV-1, suggesting a second mechanism of action. Development of resistance to ADS-J1 on the polyanion-resistant HIV-1 led to mutations in gp120 coreceptor binding site and not in gp41. Time of addition experiments confirmed that ADS-J1, but not polyanions such as dextran sulfate or AR177, worked at a step that mimics the activity of an HIV coreceptor antagonist but prior to gp41-dependent fusion. We conclude that ADS-J1 may bind to the HIV coreceptor binding site as its mechanism of anti-HIV activity.

Removal of human immunodeficiency virus by an 0.04-micron membrane filter.

We tested the ability of a 0.04-micron nylon membrane filter to remove human immunodeficiency virus (HIV) from tissue culture media containing 10% fetal calf serum. Endpoint titrations of infectious virus (ID50) were performed on lymphocyte cultures. The presence of virus in the cultures was determined using an enzyme-linked immunoabsorbant assay (ELISA) of the HIV-1 P24 core antigen. In repeated experiments, filtration of the virus suspension resulted in the removal of HIV below detectable limits. The titer reduction was estimated to be greater than 8.5 x 10(2). These results suggest that this filter is effective in removing HIV from fluids containing serum or serum products.

Detection of p24 in HIV-1 infected cells embedded in LR White and Lowicryl K4M.

In this study we present a postembedding on-grid immunogold labelling procedure for the ultrastructural localization of the HIV-1 core protein p24. HIV-1 infected cells were fixed in 0.1% glutaraldehyde, incompletely dehydrated and embedded in LR White or in Lowicryl K4M. Antigenic sites were detected by incubation of ultrathin sections with primary mouse monoclonal antibody anti-HIV-1 p24, followed by the secondary antibody goat anti-mouse IgG coupled to 10nm gold particles. Antigenicity of p24 was found to withstand the applied fixation and was shown to be preserved in LR White as well as in Lowicryl. The described procedure permits the uncomplicated and easy detection of p24 in HIV-1 infected cells and tissues.