Development of scalable manufacturing process and GMP-compatible formulation for a novel recombinant schistosomiasis vaccine

Schistosomiasis is a parasitic disease spread by fresh water snails. After malaria, schistosomiasis is the deadliest parasitic disease, plaguing an estimated 200 million people worldwide, and causing up to 280,000 fatalities in Africa. This neglected tropical disease has also emerged as an important co-factor in Africa’s HIV/AIDS epidemic, especially among women and adolescent girls. Together with hookworm disease and leishmaniasis, it ranks as the neglected tropical disease with the highest disease burden as defined by disability-adjusted life years (DALYs)1. Although treatments exist, such as praziquantel chemotherapy, a vaccine will likely be needed to prevent infection and re-infection, interrupt disease transmission, and ultimately establish long-term control and elimination of the disease. No such vaccine currently exists, but a promising candidate is currently under development at Texas Children’s Hospital Center for Vaccine Development (TCH-CVD). The Sm-TSP-2 schistosomiasis vaccine comprises a 9 kDa recombinant protein corresponding to the extracellular domain of a unique S. mansoni tetraspanin found in the parasite’s tegumental surface. Sm-TSP-2 was expressed as a recombinant protein secreted by the yeast PichiaPinkTM and purified in a two-step process, which resulted in a protein recovery yield of 31% and a protein purity of 97%.2 The developed processes were suitable for production of purified protein for subsequent formulation and Phase 1 clinical studies. However, improvements in process yield and efficiency, as well as transition of the formulation to GMP-compatible materials, are desirable for the advancement of this candidate through subsequent clinical phases and large-scale manufacturing. TCH-CVD and MilliporeSigma are conducting a collaborative project to optimize the efficiency and scalability of the Sm-STP-2 schistosomiasis vaccine manufacturing process. The overall goal of this work is to develop a safe and low-cost process for the purification of the vaccine antigen. This was accomplished by redesigning the original process, which utilized 750kD hollow fiber and 3kD cellulose membrane tangential flow filtration (TFF) devices for the clarification and concentration of the Pichia pastoris-based vaccine lysate process feed. The level of solids in the fermentation broth (30%) had required a dilution to enable processing through the hollow fiber device, which led to decreased product yield and increased complexity. MilliporeSigma assisted TCH-CVD with studies to eliminate the dilution prior to lysate clarification and to also streamline the process to enable Texas Children’s to simultaneously clarify and concentrate the yeast lysate. 0.1um, open-channel, stacked plate, membrane sheet TFF devices were successfully used to clarify the undiluted lysate. The TFF operating parameters (specifically the feed and permeate flow rates) were optimized to enable the downstream 3kD concentration process to run concurrently in a cascade TFF. Enhancement of the chromatography operations is currently underway as well. This presentation will detail the optimized clarification and concentration process and highlight the economic and process simplification benefits. Work was also performed to optimize the vaccine formulation. Extensive studies were previously conducted to identify excipients and conditions to maximize the stability of soluble recombinant Sm-TSP-2.3 Additional components were tested to find alternative GMP-grade reagents that maintained or improved the stability of the vaccine. REFRENCES 1. Forgotten People, Forgotten Diseases: The Neglected Tropical Diseases and Their Impact on Global Health and Development, Second Edition. Peter J. Hotez. American Society for Microbiology Press, Washington, DC, USA, 2013. 2. Curti E et al. Hum Vaccin Immunother. 2013 Nov;9(11):2342-50. 3. Cheng W et al. Hum Vaccin Immunother. 2013 Nov;9(11):2351-61

DETEKSI GLUKOSA DALAM URIN ORANGUTAN SUMATERA (PONGO ABELII) MENGGUNAKAN STRIPTEST SEMIKUANTITATIF DI TAMAN HEWAN PEMATANG SIANTAR

Penelitian ini bertujuan mengetahui ada tidaknya glukosa dalam uron orangutan sumatera (Pongo abelii) sebagai penunjang diagnosa di Taman Hewan Pematang Siantar, Sumatera Utara. Pengoleksian urin terhadap 4 ekor orangutan sumatera di dalam kandang yang dilakukan pada pagi hari yaitu saat orangutan bangun tidur atau sebelum pemberian pakan orangutan. Pengulangan uji dilakukan 3 kali selama 10 hari pada bulan Januari 2015. Setelah pengoleksian urin kemudian dilakukan pemeriksaan dengan cara mencelupkan stripstest pada 5-10 ml urin selama 30 detik. Analisis data menggunakan metode deskriptif kualitatif dengan hasil bersifat semikuantitatif melalui pembacaan nilai glukosa pada stripstest yang memiliki skala perubahan warna yaitu : negatif, positif 1 (100 mg/dL), positif 2 (250 mg/dL), positif 3 (500 mg/dL), dan positif 4 (1000 mg/dL). Hasil penelitian menunjukkan bahwa dari 4 sampel urin orangutan sumatera tidak terdeteksi adanya glukosa dalam urin

Real-Time Monitoring of Solid-Phase PCR Using Fiber-Optic SPR

Real-time monitoring of solid-phase DNA amplification using a surface plasmon resonance sensor (SPR) with gold nanobead signal enhancement is presented. The sensor is applied in the quantification of a DNA aptamer and subsequent melting curve analysis. The system is suited for application in medical and food-safety diagnostics, and for the study and optimization of solid-phase polymerase chain reaction. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.status: publishe

Peanut allergen detection with fiber optic SPR

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Pseudoviruses, a safer toolbox for vaccine development against enveloped viruses

ABSTRACTIntroduction Pseudoviruses are recombinant, replication-incompetent, viral particles designed to mimic the surface characteristics of native enveloped viruses. They are a safer, and cost-effective research alternative to live viruses. With the potential emergence of the next major infectious disease, more vaccine scientists must become familiar with the pseudovirus platform as a vaccine development tool to mitigate future outbreaks.Areas Covered This review aims at vaccine developers to provide a basic understanding of pseudoviruses, list their production methods, and discuss their utility to assess vaccine efficacy against enveloped viral pathogens. We further illustrate their usefulness as wet-lab simulators for emerging mutant variants, and new viruses to help prepare for current and future viral outbreaks, minimizing the need for gain-of-function experiments with highly infectious or lethal enveloped viruses.Expert opinion With this platform, researchers can better understand the role of virus–receptor interactions and entry in infections, prepare for dangerous mutations, and develop effective vaccines

Fiber optic based surface plasmon aptasensor

In this research, we aimed to develop a cost-effective surface plasmon resonance (SPR) probe for the label-free detection of human Immunoglobulin E (hIgE). This target protein plays an important role in many allergic reactions in the human body. To the best of our knowledge, this is the first research project where a SPR probe is combined with the aptamer-technology. SPR is probably the best known method for online detection of biological binding reactions. In the search for more convenient SPR-sensors, we have replaced the prism based systems by a fiber optic design. In the setup, replaceable and interchangeable sensing probes can be affixed onto the end of a bifurcated optical fiber. The fiber probe is attached to a computer-controlled robot. Aptamers are nucleic acids which have the potential to bind to specific target molecules. These selective oligonucleotides are produced through a selection process based on systematic evolution of ligands by the exponential enrichment (SELEX). Their most pronounced advantages over antibodies are stability to biodegradation and low production cost. We utilized this high stability of the DNA aptamers to develop reusable biosensors. The gold surface of the sensor is coated with a mixture of mercapto poly-ethylene glycol and different length mercapto alcohol chains. On this layer, streptavidin is immobilized by 1-Ethyl-3-[3-dimethyl aminopropyl]carbodiimide hydrochloride. Finally, biotinylated aptamers are attached. Our results illustrate that this surface design prevents non-specific binding and is very stable, which is important for regeneration. The sensors have an average detection limit of 2 nM for hIgE. The detection range depends on the amount of aptamers immobilized on the sensor surface. We are able to regenerate the aptasensors for more than 25 measurements. No wavelength shift was visible when the same experiment was repeated with hIgG. In the near future we will repeat these experiments in real blood serum samples.status: publishe

SURFACE PLASMON RESONANCE APTA-SENSOR FOR FOOD ALLERGEN DETECTION

status: publishe

Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices

The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE)-SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind with other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, which are surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 ± 82 nM, 419 ± 63 nM, and 450 ± 60 nM, respectively). Furthermore, the selected aptamer was used for bioassay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein.publisher: Elsevier articletitle: Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices journaltitle: Biosensors and Bioelectronics articlelink: http://dx.doi.org/10.1016/j.bios.2012.12.022 content_type: article copyright: Copyright © 2012 Elsevier B.V. All rights reserved.status: publishe

Real-time monitoring of DNA hybridization and melting processes using a fiber optic sensor

In this paper a fiber optic surface plasmon resonance (FO-SPR) sensor was used to analyze the melting process of DNA linked to silica nanoparticles. Real-time monitoring of a DNA melting process has rarely been studied using surface plasmon resonance (SPR), since most commercial SPR setups do not allow for dynamic and accurate temperature control above 50 °C. The FO-SPR sensor platform, with silica nanobead signal amplification, allows sensing inside a standard PCR thermocycler, which makes high resolution DNA melting curve analysis possible. This innovative combination was used to characterize the hybridization and melting events between DNA immobilized on the sensor surface and DNA probes on silica nanoparticles. At optimized hybridization conditions complementary DNA strands of different length could be distinguished. While the real-time FO-SPR analysis of DNA hybridization did not result in significant variances, the analysis of DNA melting determined the exact length of overlap and the matching Gibbs energy.status: publishe