Lipoprotein capture ELISA method for the sensitive detection of amphiphilic biomarkers
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59 BASIC BIOLOGICAL SCIENCES↗
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Overview of Rotor Hover Performance Capabilities at Low Reynolds Number for Mars Exploration
The Evolutionary aLgorithm for Iterative Studies of Aeromechanics (ELISA) software was developed in support of the Rotorcraft Optimization for the Advancement of Mars eXploration (ROAMX) project. ELISA was developed to enable aerodynamic rotor hover optimization for low Reynolds number flows in the Mars atmosphere. ELISA comprises two modules. The first module is dedicated to airfoil optimization and allows for the creation of multi-objective Pareto optimal (PO) airfoil sets with the airfoil performance evaluation performed using OVERFLOW. The second module is dedicated to rotor hover performance optimization and generates multi-objective PO rotor sets with the rotor performance evaluation performed using the comprehensive analysis code CAMRAD II. This paper presents recent updates to the ELISA optimization toolset. The airfoil module now includes variation in section Reynolds number, alongside simultaneous maximization of section lift and minimization of section drag. Consequently, the rotor optimization module can query PO airfoil sets (as a function of section lift, drag, and Reynolds number) and generate PO C81 decks tailored to specific Reynolds numbers, eliminating the need for adequate initial chord guesses and allowing for arbitrary rotor solidities to be studied. Furthermore, the rotor optimization has been extended to incorporate a third dimension, alongside maximization of blade loading and minimization of rotor power. This enables optimization across a relevant density range on Mars, presenting the lowest power rotor hover geometry, for each attainable blade loading, for each density. The goal of this work is to present the relevance of recent updates to the ELISA optimization toolset, by showing full rotor hover optimization using unconventional airfoils across a practical Mars density range, and by presenting various optimizations for changing blade numbers with unconstrained solidity in the Mars atmosphere.
Rotor↗
Persistent Seroconversion after Accidental Eye Exposure to Calcifying Nanoparticles
Biosafety of nanomaterials has attracted much attention recently. We report here a case where accidental human eye exposure to biogenic nanosized calcium phosphate in the form of calcifying nanoparticles (CNP) raised a strong IgG immune response against proteins carried by CNP. The antibody titer has persisted over ten years at the high level. The IgG was detected by ELISA using CNPs propagated in media containing bovine and human serum as antigen. The exposure incident occurred to a woman scientist (WS) at a research laboratory in Finland at 1993. CNP, also termed "nanobacteria", is a unique self-replicating agent that has not been fully characterized and no data on biohazards were available at that time. Before the accident, her serum samples were negative for both CNP antigen and anti-CNP antibody using specific ELISA tests (Nanobac Oy, Kuopio, Finland). The accident occurred while WS was harvesting CNP cultures. Due to a high pressure in pipetting, CNP pellet splashed into her right eye. Both eyes were immediately washed with water and saline. The following days there was irritation and redness in the right eye. These symptoms disappeared within two weeks without any treatment. Three months after the accident, blood and urine samples of WS were tested for CNP cultures (2), CNP-specific ELISA tests, and blood cell counts. Blood cell counts were normal, CNP antigen and culture tests were negative. A high IgG anti-CNP antibody titer was detected (see Figure). The antibodies of this person have been used thereafter as positive control and standard in ELISA manufacturing (Nano-Sero IgG ELISA, Nanobac Oy, Kuopio, Finland).
Ciftcioglu, Neva↗
Technology Development Roadmap: A Technology Development Roadmap for a Future Gravitational Wave Mission
Humankind will detect the first gravitational wave (GW) signals from the Universe in the current decade using ground-based detectors. But the richest trove of astrophysical information lies at lower frequencies in the spectrum only accessible from space. Signals are expected from merging massive black holes throughout cosmic history, from compact stellar remnants orbiting central galactic engines from thousands of close contact binary systems in the Milky Way, and possibly from exotic sources, some not yet imagined. These signals carry essential information not available from electromagnetic observations, and which can be extracted with extraordinary accuracy. For 20 years, NASA, the European Space Agency (ESA), and an international research community have put considerable effort into developing concepts and technologies for a GW mission. Both the 2000 and 2010 decadal surveys endorsed the science and mission concept of the Laser Interferometer Space Antenna (LISA). A partnership of the two agencies defined and analyzed the concept for a decade. The agencies partnered on LISA Pathfinder (LPF), and ESA-led technology demonstration mission, now preparing for a 2015 launch. Extensive technology development has been carried out on the ground. Currently, the evolved Laser Interferometer Space Antenna (eLISA) concept, a LISA-like concept with only two measurement arms, is competing for ESA's L2 opportunity. NASA's Astrophysics Division seeks to be a junior partner if eLISA is selected. If eLISA is not selected, then a LISA-like mission will be a strong contender in the 2020 decadal survey. This Technology Development Roadmap (TDR) builds on the LISA concept development, the LPF technology development, and the U.S. and European ground-based technology development. The eLISA architecture and the architecture of the Mid-sized Space-based Gravitational-wave Observatory (SGO Mid)-a competitive design with three measurement arms from the recent design study for a NASA-led mission after 2020-both use the same technologies. Further, NASA participation in an ESA-led mission would likely augment the eLISA architecture with a third arm to become the SGO Mid architecture. For these reasons, this TDR for a future GW mission applies to both designs and both programmatic paths forward. It is adaptable to the different timelines and roles for an ESA-led or a NASA-led mission, and it is adaptable to available resources. Based on a mature understanding of the interaction between technology and risk, the authors of this TDR have chosen a set of objectives that are more expansive than is usual. The objectives for this roadmap are: (1) reduce technical and development risks and costs; (2) understand and, where possible, relieve system requirements and consequences; (3) increase technical insight into critical technologies; and (4) validate the design at the subsystem level. The emphasis on these objectives, particularly the latter two, is driven by outstanding programmatic decisions, namely whether a future GW mission is ESA-led or NASA-led, and availability of resources. The relative emphasis is best understood in the context of prioritization.
Laser Interferometry Space Antenna↗
Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics
ABSTRACT California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA. Importance Orthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.
Microbiology↗
From glomalin to glomalose: unraveling the molecular identity of the MAb32B11 antigen
Glomalin, a substance produced by arbuscular mycorrhizal (AM) fungi, haswell-documented benefits for plant and soil health, including water retention and soil aggre-gation. Glomalin quantification has been performed by enzyme-linked immunosorbent assay(ELISA) using a monoclonal antibody, MAb32B11, that has been described as targeting a heatshock protein 60 (RiHSP60). In this study, we re-examined the molecular nature of the antigen recognized byMAb32B11. MAb32B11 did not cross-react with the RiHSP60 polypeptide. Glomalin extracts of Rhizo-phagus irregularis showed strong and dose-dependent cross-reactivity with MAb32B11 evenwhen protein levels were undetectable, raising doubts about the proteinaceous nature of theantigen. Protease treatments of glomalin extracts did not affect the ELISA signal. However,treatment with periodate, which degrades polysaccharides, significantly reduced the signal. Astrong correlation between carbohydrate content and the ELISA signal was observed in glo-malin extracts. These findings indicate that MAb32B11 recognizes a carbohydrate, likely originating fromcell walls of AM fungi. Further analysis of glomalin extracts using size exclusion chromatogra-phy suggests that the epitope of MAb32B11 is a complex carbohydrate in the size range of511–600 kDa. Understanding the true nature of glomalin will enhance our ability to quantifyit accurately and leverage its agricultural benefits.
59 BASIC BIOLOGICAL SCIENCES↗
Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means
Abstract Background Type I interferons are widely used in research applications and as biotherapeutics. Current assays used to measure interferon concentrations, such as plaque reduction assays and ELISA, are expensive, technically challenging, and may take days to provide results. We sought to develop a robust and rapid assay to determine interferon concentrations produced from transiently transfected cell cultures. Method Indirect quantification of recombinant interferon was evaluated using a novel bi-cistronic construct encoding the Foot-and-mouth disease virus 2A translational interrupter sequence to yield equimolar expression of Gaussia princeps luciferase and porcine interferon α. Direct quantification was evaluated by expression of a novel fusion protein comprised of Gaussia princeps luciferase and porcine type I interferon. Plasmids encoding constructs are transiently transfected into cell cultures and supernatant harvested for testing of luminescence, ELISA determined concentration, and anti-viral activity against vesicular stomatitis virus. Results Bi-cistronic constructs, utilized for indirect quantification, demonstrate both luciferase activity and anti-viral activity. Fusion proteins, utilized for direct quantification, retained secretion and luminescence however only the interferon α fusion protein had antiviral activity comparable to wildtype porcine interferon α. A strong linear correlation was observed between dilution and luminescence for all compounds over a dynamic range of concentrations. Conclusion The correlation of antiviral and luciferase activities demonstrated the utility of this approach, both direct and indirect, to rapidly determine recombinant interferon concentrations. Concentration can be determined over a more dynamic concentration range than available ELISA based assays using this methodology.
59 BASIC BIOLOGICAL SCIENCES↗
RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer
Chronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrP Sc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer. A total of 23 platelet samples derived from CWD-positive deer (ELISA +/IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10 -2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10 -3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels. Incorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component signifcantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.
59 BASIC BIOLOGICAL SCIENCES↗
Single-Atom Nanozymes Linked Immunosorbent Assay for Sensitive Detection of A β 1-40: A Biomarker of Alzheimer’s Disease
Single-atom nanozymes (SANs) possess unique features of maximum atomic utilization and present highly assembled enzyme-like structure and remarkable enzyme-like activity. By introducing SANs into immunoassay, limitations of ELISA such as low stability of horseradish peroxidase (HRP) can be well addressed, thereby improving the performance of the immunoassays. In this work, we have developed novel Fe-N-C single-atom nanozymes (Fe-N x SANs) derived from Fe-doped polypyrrole (PPy) nanotube and substituted the enzymes in ELISA kit for enhancing the detection sensitivity of amyloid beta 1-40. Results indicate that the Fe-N x SANs contain high density of single-atom active sites and comparable enzyme-like properties as HRP, owing to the maximized utilization of Fe atoms and their abundant active sites, which could mimic natural metalloproteases structures. Further designed SAN-linked immunosorbent assay (SAN-LISA) demonstrates the ultralow limit of detection (LOD) of 0.88 pg/mL, much more sensitive than that of commercial ELISA (9.98 pg/mL). The results confirm that the Fe-N x SANs can serve as a satisfactory replacement of enzyme labels, which show great potential as an ultrasensitive colorimetric immunoassay.
60 APPLIED LIFE SCIENCES↗
A solid phase enzyme-linked immunosorbent assay for the antigenic detection of Legionella pneumophila (serogroup 1): A compliment for the space station diagnostic capability
It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events and environmental monitoring during long periods of space flight. The application of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be facilitated through employment of serological methods to aid in the identification of bacterial, fungal, and viral agents. A number of serological approaches are currently being considered, including the use of Enzyme Linked Immunosorbent Assay (ELISA) technology, which could be utilized during microgravity conditions. A solid phase, membrane supported ELISA for the detection of Legionella pneumophila, an expected disease agent, was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. These studies demonstrate the capability of membrane supported ELISA systems for identification of expected microbial disease agents as part of the HMF.
Hejtmancik, Kelly E.↗
Calbindins decreased after space flight
Exposure of the body to microgravity during space flight causes a series of well-documented changes in Ca2+ metabolism, yet the cellular and molecular mechanisms leading to these changes are poorly understood. Calbindins, vitamin D-dependent Ca2+ binding proteins, are believed to have a significant role in maintaining cellular Ca2+ homeostasis. In this study, we used biochemical and immunocytochemical approaches to analyze the expression of calbindin-D28k and calbindin-D9k in kidneys, small intestine, and pancreas of rats flown for 9 d aboard the space shuttle. The effects of microgravity on calbindins in rats from space were compared with synchronous Animal Enclosure Module controls, modeled weightlessness animals (tail suspension), and their controls. Exposure to microgravity resulted in a significant and sustained decrease in calbindin-D28k content in the kidney and calbindin-D9k in the small intestine of flight animals, as measured by enzyme-linked immunosorbent assay (ELISA). Modeled weightlessness animals exhibited a similar decrease in calbindins by ELISA. Immunocytochemistry (ICC) in combination with quantitative computer image analysis was used to measure in situ the expression of calbindins in the kidney and the small intestine, and the expression of insulin in pancreas. There was a large decrease of immunoreactivity in renal distal tubular cell-associated calbindin-D28k and in intestinal absorptive cell-associated calbindin-D9k of space flight and modeled weightlessness animals compared with matched controls. No consistent difference in pancreatic insulin immunoreactivity between space flight, modeled weightlessness, and controls was observed. Regression analysis of results obtained by quantitative ICC and ELISA for space flight, modeled weightlessness animals, and their controls demonstrated a significant correlation. These findings after a short-term exposure to microgravity or modeled weightlessness suggest that a decreased expression of calbindins may contribute to the disorders of Ca2+ metabolism induced by space flight.
Non-NASA Center↗
A Preliminary Analysis of Calcifying Particles in the Serum and Prostates of Patients with Prostatic Inflammation
Chronic diseases of the prostate such as benign prostatic hyperplasia (BPH) & chronic pelvic pain syndrome (CPPS) have associated findings of chronic inflammation, despite a lack of causal relationship. Numerous attempts to define an infectious agent responsible for the clinical findings have been inconsistent. The possibility of an infectious agent, that has not been uncovered with routine culturing methods, forms the basis for this study. Serum from 940 healthy Finnish men were compared with serum from 40 Crohn's, 40 path dx prostatitis, & 40 with path dx carcinoma, using an enzyme-linked immunosorbant assay (ELISA), to detect antigens specific to Nanobacteria(NB) utilizing monoclonal antibodies (Ab) 5/3 and 8D10. This ELISA has not been validated for detecting NB-associated with clinical prostatic disease, yet cross-reactivity with other bacterial species is low. Immunohistochemistry was performed on de-paraffinized prostatic tissue slides, de-calcified with EDTA and stained with the DAKO Catalyzed Signal Amplification kit, employing 8D10 as the primary (target/antigen-detecting) Ab. The mean (plus or minus SD) & median concentrations of NB antigen (U/50 L) were 379.59 (plus or minus 219.28) & 640.00 for patients with prostatitis (BPH) vs 3.31 (plus or minus 3.55) & 2.94 for prostate adenocarcinoma, 1.88 (plus or minus 2.94) & 0.80 for Crohn's disease, & 7.43 (plus or minus 25.57) & 0.00 for patients with no clinical prostatic disease. Unpaired t-tests revealed statistically significant differences between the prostatitis (BPH) sera & each of the other groups with p less than 0.005, but no differences between the other groups themselves. Preliminary studies with immunohistochemistry & 3-D confocal microscopy reveal 16/24 tissue sections + for NB Ag in BPH vs. only 2/22 tissue sections with prostate cancer. The preliminary findings of this serum screening study suggest that NB antigen may be commonly found in the serum of patients with the pathological diagnosis of prostatitis. Preliminary immunohistologic studies, suggest that NB may be found within the gland itself at a higher rate in patients with BPH relative to patients with adenocarcinoma, however confirmatory studies with a more specific ELISA technique, primary cultures, & with larger numbers of patients in a prospective design are required to determine if 1) NB are a causative organism for clinical hyperplastic and inflammatory disease, & if 2) serological testing can be used to discriminate patients with nanobacterial-associated prostatic disease.
Jones, Jeffrey A.↗
Gravitational-wave Mission Study
In November 2013, ESA selected the science theme, the "Gravitational Universe," for its third large mission opportunity, known as L3, under its Cosmic Vision Programme. The planned launch date is 2034. ESA is considering a 20% participation by an international partner, and NASA's Astrophysics Division has indicated an interest in participating. We have studied the design consequences of a NASA contribution, evaluated the science benefits and identified the technology requirements for hardware that could be delivered by NASA. The European community proposed a strawman mission concept, called eLISA, having two measurement arms, derived from the well studied LISA (Laser Interferometer Space Antenna) concept. The US community is promoting a mission concept known as SGO Mid (Space-based Gravitational-wave Observatory Mid-sized), a three arm LISA-like concept. If NASA were to partner with ESA, the eLISA concept could be transformed to SGO Mid by the addition of a third arm, augmenting science, reducing risk and reducing non-recurring engineering costs. The characteristics of the mission concepts and the relative science performance of eLISA, SGO Mid and LISA are described. Note that all results are based on models, methods and assumptions used in NASA studies
identified the technology requirements↗
Experimental Results for Mars Rotorcraft Airfoils (roamx-0201 and clf5605) at Low Reynolds Number and Compressible Flow in a Mars Wind Tunnel
Experimental results are obtained for a roamx-0201 type airfoil and the clf5605 airfoil at highsubsonic, low Reynolds number conditions using the Tohoku University Mars Wind Tunnel, Japan. The tests are conducted at a Mach number of M = 0.60, and a Reynolds number of Re = 20,000 to reflect representative aerodynamics of a rotor blade for Mars exploration. The angle of attack is varied between α = −2.0 deg and α = 6.0 deg. The roamx-0201 type airfoil is an unconventional airfoil optimized for the chosen tunnel operating conditions using the Evolutionary aLgorithm for Iterative Studies of Aeromechanics (ELISA), developed under the Rotor Optimization for the Advancement of Mars eXploration (ROAMX) project. ELISA is utilized here to optimize aerodynamic airfoil performance using a Genetic Algorithm and two-dimensional high-fidelity CFD simulations, ultimately resulting in a Pareto-optimal airfoil set. The clf5605 airfoil is the outboard airfoil used on the Ingenuity Mars Helicopter and provides a baseline against which the roamx-0201, as well as possible future airfoil profiles for the compressible low Reynolds number regime, can be compared against. Lift and drag data are recorded using a balance, pressure distributions are obtained using Pressure Sensitive Paint (PSP) application, and Schlieren images are obtained to visualize the flowfield. The data is tabulated to aid future research.
Roamx↗
High-Sensitivity DNA Aptasensors for Detecting Salivary Biomarker S100A7 in Heart Failure
Early detection of heart failure (HF) is vital for improving patient outcomes, lowering hospital readmission rates, and enabling prompt treatment. We present the first high-affinity DNA aptamer for the salivary HF biomarker S100A7 and its application in highly sensitive, noninvasive diagnostic tests. Iterative truncation of the initial 82-nt aptamer (17–82) produced a 43-nt core (17–43) with a binding affinity of 27 nM, which was further enhanced to 5.5 nM through dimerization. Biochemical and mutational studies confirmed that 17–43 adopts a G-quadruplex structure, which is essential for S100A7 recognition and resistance to enzymatic degradation in human saliva. Incorporating 17–43 into sandwich aptamer-ELISA and hybrid aptamer–antibody ELISA assays allowed detection of recombinant S100A7 in human saliva with limits of detection (LOD) of 7.4 ng mL–1 (0.6 nM) and 29 pg/mL (2.2 pM), respectively-outperforming commercial immunoassays in both sensitivity and dynamic range. The hybrid assay maintained its full performance after 2.5 months of room temperature storage. Additionally, a biolayer interferometry (BLI) sensor with 17–43 quantified S100A7 in patient saliva (n = 3), achieving a LOD of 3.2 ng mL–1 (0.3 nM) with a total assay time of less than 20 min. The aptamer’s stability, high specificity, and versatility across biosensing platforms establish it as a promising tool for noninvasive heart failure diagnostics, laying the groundwork for portable aptamer-based biosensors for multiplexed monitoring of HF biomarkers.
DNA aptamer↗
Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate
Background: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. Methods: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. Results: Purified nHRP2 was identified by SDS-PAGE and western blot as a –60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. Conclusions: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.
59 BASIC BIOLOGICAL SCIENCES↗
Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens
The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.
60 APPLIED LIFE SCIENCES↗