Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy.

In-line monitoring of surfactant clearance in viral vaccine downstream processing.

PURPOSE: The goal of this study is to examine the suitability of in-line infrared measurements to monitor, in real-time, surfactant concentration in the viral vaccine drug substance during a 50KDa tangential flow filtration (TFF) process. METHODS: A ReactIR™ 702L instrument was used to gather spectra of process off-line samples and reference materials to assess the feasibility of monitoring surfactant concentration during a TFF process in real-time. Both univariate and multivariate models were used to evaluate the off-line sample data and were found to be in good agreement with surfactant concentration values obtained by HPLC. These results were used as justification for a real-time TFF experiment with live process material. RESULTS: Small scale ReactIR experiments with process material demonstrated that a multivariate model using the 1300 cm-1 to 1000 cm-1 spectral region can be used to predict surfactant concentrations between TFF exchanges 8 to 15. CONCLUSION: The results of this study demonstrated suitability of an in-line infrared measurement to monitor surfactant concentration in the viral vaccine drug substance between exchanges 8-15 of a 50 kDa tangential flow filtration process. The preliminary multivariate model used for this work can be further optimized for the in-line use at manufacturing scale.

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization "Hotspots" in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction.

The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these "binding-site adjacent" dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these "peripheral" sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization "hotspots" outside of the core binding interfaces defined by high resolution structural data.

PAT solutions to monitor adsorption of Tetanus Toxoid with aluminum adjuvants.

The focus of this study was to examine the small-scale adsorption process of Tetanus Toxoid (TT) as a model protein antigen to aluminum phosphate (AlPO4) and aluminum oxyhydroxide (AlOOH) adjuvants with real-time monitoring by in-line ReactIR™, ParticleTrack™ based on Focused Beam Reflectance Measurement (FBRM) and EasyViewer™ probes. The adsorption process of AlPO4 and AlOOH with TT using was monitored in the small-scale reactors. Conformational changes in TT were monitored using in-line infrared probe ReactIR, whereas particle formation associated with protein adsorption were measured by particle size, count, and imaging tools, such as ParticleTrack with FBRM and EasyViewer probes. ParticleTrack distribution results and kinetic measurements were also supported by observations made using EasyViewer. In addition to EasyMax, BioBLU reactor was also used for the adsorption experiments. ReactIR with ATR-Fiber probe was effectively able to monitor adsorption progress of TT to AlOOH and to AlPO4. ReactIR, EasyViewer, and ParticleTrack provided detailed mechanistic and kinetic information for reaction of TT with AlPO4 and AlOOH. These in-situ measurements revealed a possible multi-step process for TT to AlPO4 which may be an indication of antigen adsorption.

Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-Line and In-Line Tools.

PURPOSE: Aluminum-based adjuvants including aluminum phosphate (AlPO4) are commonly used in many human vaccines to enhance immune response. The interaction between the antigen and adjuvant, including the physical adsorption of antigen, may play a role in vaccine immunogenicity and is a useful marker of vaccine product quality and consistency. Thus, it is important to study the physicochemical properties of AlPO4, such as particle size and chemical composition. Control of the vaccine adjuvant throughout the manufacturing process, including raw materials and the intermediate and final product stages, can be effectively achieved through monitoring of such key product attributes to help ensure product quality. METHODS: This study focuses on the compositional analysis of AlPO4 adjuvant at the intermediate and final manufacturing stages using the off-line methods Fourier-Transform Infrared (FTIR) and Raman spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and the in-line method Attenuated Total Reflectance (ATR). Particle size distribution of AlPO4 was measured off-line using Laser diffraction (LD) and in-line using Focused Beam Reflectance Measurement (FBRM®). RESULTS: There was no observable difference in size distribution between the intermediate and final stage AlPO4 by off-line and in-line analysis, in both small- or large-scale production samples. Consistent peak shifts were observed in off-line and in-line infrared (IR) spectroscopy as well as off-line XPS for both small- and large-scale AlPO4 manufacturing runs. Additionally, IR spectroscopy and FBRM® for size distribution were used as in-line process analytical technology (PAT) to monitor reaction progress in real-time during small-scale AlPO4 manufacturing from raw materials. The small-scale adsorption process of a model protein antigen (Tetanus toxoid) to AlPO4 adjuvant was also monitored by in-line ReactIR probe. CONCLUSION: This study demonstrated that in-line PAT can be used to monitor particle size and chemical composition for the various stages of adjuvant manufacturing from raw materials through intermediate to final adjuvant product stage. Similar approaches can be utilized to help assess lot-to-lot consistency during adjuvant manufacturing and vaccine product development. Moreover, the use of in-line PAT is highly conductive to advanced manufacturing strategies such as real-time product release testing and automated processes of the future.

A novel approach to neutron dosimetry.

PURPOSE: Having been overlooked for many years, research is now starting to take into account the directional distribution of neutron workplace fields. Existing neutron dosimetry instrumentation does not account for this directional distribution, resulting in conservative estimates of dose in neutron workplace fields (by around a factor of 2, although this is heavily dependent on the type of field). This conservatism could influence epidemiological studies on the health effects of radiation exposure. This paper reports on the development of an instrument which can estimate the effective dose of a neutron field, accounting for both the direction and the energy distribution. METHODS: A 6Li-loaded scintillator was used to perform neutron assays at a number of locations in a 20 × 20 × 17.5 cm3 water phantom. The variation in thermal and fast neutron response to different energies and field directions was exploited. The modeled response of the instrument to various neutron fields was used to train an artificial neural network (ANN) to learn the effective dose and ambient dose equivalent of these fields. All experimental data published in this work were measured at the National Physical Laboratory (UK). RESULTS: Experimental results were obtained for a number of radionuclide source based neutron fields to test the performance of the system. The results of experimental neutron assays at 25 locations in a water phantom were fed into the trained ANN. A correlation between neutron counting rates in the phantom and neutron fluence rates was experimentally found to provide dose rate estimates. A radionuclide source behind shadow cone was used to create a more complex field in terms of energy and direction. For all fields, the resulting estimates of effective dose rate were within 45% or better of their calculated values, regardless of energy distribution or direction for measurement times greater than 25 min. CONCLUSIONS: This work presents a novel, real-time, approach to workplace neutron dosimetry. It is believed that in the research presented in this paper, for the first time, a single instrument has been able to estimate effective dose.

Doxorubicin and 5-fluorouracil induced accumulation and transcriptional activity of p53 are independent of the phosphorylation at serine 15 in MCF-7 breast cancer cells.

The chemotherapeutic agents doxorubicin (dox) or 5-fluorouracil (5FU) are used to treat cancer cells as they cause irreparable DNA damage, inducing these aberrant cells to undergo cell death. The mediator of this process is presumed to be in part the tumor suppressor p53 which regulates genes involved in DNA repair and cell death. When MCF-7 breast cancer cells are treated with these drugs, we observed that the level of p53 and the p53 negative regulator, Mdm2, increased, as seen by others. But contrary to some reports, we observed minimal phosphorylation of p53 at serine 15 in MCF-7 cells after drug treatment. Interestingly, we determined that there was differential regulation of the kinases ATM and Chk2 with the drug treatments, likely the cause for the lack of phosphorylation of p53. We found a dramatic drop in p53 DNA binding affinity for p21 and other gene response elements (RE) after drug treatment. To determine if the p53 that accumulated in the drug treated cells was functionally active, we monitored changes in the protein products of two p53-regulated genes following drug treatment with and without the addition of a p53-specific siRNA. In response to 5FU, both p21 and Mdm2 proteins increased and that increase was alleviated if a p53-specific siRNA was added. This effect was not seen with the addition of dox. Thus, the phosphorylation at serine 15 is not necessary for the functional activation of this transcription factor. We propose a new model for the regulation of p53, Mdm2, and MdmX after drug treatment.