11 research outputs found
Application of analytical characterization tools in process and formulation development of low cost vaccines using the ULTRA manufacturing platform
In 2016, an estimated 19.5 million infants worldwide did not receive routine life-saving vaccinations according to the World Health Organization (WHO)1. Two major limitations in improving global vaccination coverage include the costs associated with vaccine manufacturing and the challenges associated with maintaining a consistent supply. The aim of the ‘ULTRA’ project (Ultra Low-cost TRansferable Automated Platform for Vaccine Manufacturing) is to standardize the development and production of new protein subunit vaccine candidates at globally affordable costs by creating a generic, low-cost, integrated, and automated vaccine manufacturing platform. In a collaborative effort between MIT, UCL, and KU (Figure 1), state-of-the-art analytical tools will be utilized to provide well-characterized vaccine bulk and drug product facilitating process changes and reduced QC costs.
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Concordance of in vitro and in vivo measures of non-replicating rotavirus vaccine potency
Rotavirus infections remain a leading cause of morbidity and mortality among infants residing in low- and middle-income countries. To address the large need for protection from this vaccine-preventable disease we are developing a trivalent subunit rotavirus vaccine which is currently being evaluated in a multinational Phase 3 clinical trial for prevention of serious rotavirus gastroenteritis. Currently, there are no universally accepted in vivo or in vitro models that allow for correlation of field efficacy to an immune response against serious rotavirus gastroenteritis. As a new generation of non-replicating rotavirus vaccines are developed the lack of an established model for evaluating vaccine efficacy becomes a critical issue related to how vaccine potency and stability can be assessed. Our previous publication described the development of an in vitro ELISA to quantify individual vaccine antigens adsorbed to an aluminum hydroxide adjuvant to address the gap in vaccine potency methods for this non-replicating rotavirus vaccine candidate. In the present study, we report on concordance between ELISA readouts and in vivo immunogenicity in a guinea pig model as it relates to vaccine dosing levels and sensitivity to thermal stress. We found correlation between in vitro ELISA values and neutralizing antibody responses engendered after animal immunization. Furthermore, this in vitro assay could be used to demonstrate the effect of thermal stress on vaccine potency, and such results could be correlated with physicochemical analysis of the recombinant protein antigens. This work demonstrates the suitability of the in vitro ELISA to measure vaccine potency and the correlation of these measurements to an immunologic outcome
Developability Assessment of Physicochemical Properties and Stability Profiles of HIV-1 BG505 SOSIP.664 and BG505 SOSIP.v4.1-GT1.1 gp140 Envelope Glycoprotein Trimers as Candidate Vaccine Antigens
This work is licensed under a Creative Commons Attribution 4.0 International License.The induction of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an effective vaccine against HIV-1. A soluble, trimeric, germline (gI) bNAb-targeting variant of the HIV-1 envelope glycoprotein (termed BG505 SOSIP.v4.1-GT1.1 gp140, abbreviated to GT1.1) has recently been developed. Here, we have compared this new immunogen with the parental trimer from which it was derived, BG505 SOSIP.664 gp140. We used a comprehensive suite of biochemical and biophysical methods to determine physicochemical similarities and differences between the 2 trimers, and thereby assessed whether additional formulation development efforts were needed for the GT1.1 vaccine candidate. The overall higher order structure and oligomeric states of the 2 vaccine antigens were quite similar, as were their thermal, chemical, and colloidal stability profiles, as evaluated during accelerated stability studies. Overall, we conclude that the primary sequence changes made to create the gl bNAb-targeting GT1.1 trimer did not detrimentally affect its physicochemical properties or stability profiles from a pharmaceutical perspective. This developability assessment of the BG505 GT1.1 vaccine antigen supports using the formulation and storage conditions previously identified for the parental SOSIP.664 trimer and enables the development of GT1.1 for phase I clinical studies.Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Development (OPP1147661)Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Development (OPP1153692)NIH HIVRAD grant P01 AI 110657Aids Fonds grant 201601
Daksha: On Alert for High Energy Transients
We present Daksha, a proposed high energy transients mission for the study of
electromagnetic counterparts of gravitational wave sources, and gamma ray
bursts. Daksha will comprise of two satellites in low earth equatorial orbits,
on opposite sides of earth. Each satellite will carry three types of detectors
to cover the entire sky in an energy range from 1 keV to >1 MeV. Any transients
detected on-board will be announced publicly within minutes of discovery. All
photon data will be downloaded in ground station passes to obtain source
positions, spectra, and light curves. In addition, Daksha will address a wide
range of science cases including monitoring X-ray pulsars, studies of
magnetars, solar flares, searches for fast radio burst counterparts, routine
monitoring of bright persistent high energy sources, terrestrial gamma-ray
flashes, and probing primordial black hole abundances through lensing. In this
paper, we discuss the technical capabilities of Daksha, while the detailed
science case is discussed in a separate paper.Comment: 9 pages, 3 figures, 1 table. Additional information about the mission
is available at https://www.dakshasat.in
Science with the Daksha High Energy Transients Mission
We present the science case for the proposed Daksha high energy transients
mission. Daksha will comprise of two satellites covering the entire sky from
1~keV to ~MeV. The primary objectives of the mission are to discover and
characterize electromagnetic counterparts to gravitational wave source; and to
study Gamma Ray Bursts (GRBs). Daksha is a versatile all-sky monitor that can
address a wide variety of science cases. With its broadband spectral response,
high sensitivity, and continuous all-sky coverage, it will discover fainter and
rarer sources than any other existing or proposed mission. Daksha can make key
strides in GRB research with polarization studies, prompt soft spectroscopy,
and fine time-resolved spectral studies. Daksha will provide continuous
monitoring of X-ray pulsars. It will detect magnetar outbursts and high energy
counterparts to Fast Radio Bursts. Using Earth occultation to measure source
fluxes, the two satellites together will obtain daily flux measurements of
bright hard X-ray sources including active galactic nuclei, X-ray binaries, and
slow transients like Novae. Correlation studies between the two satellites can
be used to probe primordial black holes through lensing. Daksha will have a set
of detectors continuously pointing towards the Sun, providing excellent hard
X-ray monitoring data. Closer to home, the high sensitivity and time resolution
of Daksha can be leveraged for the characterization of Terrestrial Gamma-ray
Flashes.Comment: 19 pages, 7 figures. Submitted to ApJ. More details about the mission
at https://www.dakshasat.in
Plant Disease Detection Using Cnn
Plants and crops that are infected by pests have an impact on the country's agricultural production. Usually, farmers or professionals keep a close eye on the plants in order to discover and identify diseases. However, this procedure is frequently time-consuming, costly, and imprecise. Plant disease detection can be done by looking for a spot on the diseased plant's leaves. The goal of this paper is to create a Disease Recognition Model that is supported by leaf image classification. To detect plant diseases, we are utilizing image processing with a Convolution neural network (CNN). A convolutional neural network (CNN) is a form of artificial neural network that is specifically intended to process pixel input and is used in image recognition
Crystallization of a nonreplicating rotavirus vaccine candidate
Nonreplicating rotavirus vaccine (NRRV) candidates are being developed with the aim of serving the needs of developing countries. A significant proportion of the cost of manufacturing such vaccines is the purification in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This communication describes a systematic approach for the design of the crystallization of an NRRV candidate, VP8 subunit proteins fused to the P2 epitope of tetanus toxin, using first-principles models and preliminary experimental data. The first-principles models are applied to literature data to obtain feasible crystallization conditions and lower bounds for nucleation and growth rates. Crystallization is then performed in a hanging-drop vapor diffusion system, resulting in the nucleation and growth of NRRV crystals. The crystals obtained in a scaled-up evaporative crystallization contain proteins truncated in the P2 region, but have no significant differences with the original samples in terms of antibody binding and overall conformational stability. These results demonstrate the promise of evaporative crystallization of the NRRV
Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine
© 2020 The Authors In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed
Assessment of changes in plasma hemoglobin and potassium levels in red cell units during processing and storage
Rapid Developability Assessments to Formulate Recombinant Protein Antigens as Stable, Low-Cost, Multi-Dose Vaccine Candidates: Case-Study With Non-Replicating Rotavirus (NRRV) Vaccine Antigens
© 2020 The Authors A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries