An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Enabling Lyophilized Pneumococcal Conjugate Vaccines Through Formulation Design and Excipient Selection Suitable for A Multivalent Adjuvanted Vaccine.

Despite a consistent benefit of existing pneumococcal conjugate vaccine (PCV) on invasive pneumococcal disease and pneumonia across different epidemiological settings a tremendous gap exists towards global PCV coverage. Currently, no lyophilized dosage form exists in the PCV global vaccine marketplace and currently licensed vaccines target some, but not all relevant serotypes of Streptococcus pneumoniae. The development of lyophilized presentations of an adjuvanted multivalent vaccine formulation that aligns with the evolving epidemiological assessment of the pneumococcal disease offers broader coverage with distinct cold chain and thermostability advantages. To make progress towards this goal, we evaluated the feasibility of developing new formulation to enable a lyophilized adjuvanted PCV vaccine containing 15 different serotypes. Our findings successfully demonstrate a formulation design space that enables enhanced physical stability which controls vaccine agglomeration, preserves in-vitro vaccine potency, maintains PCV antigen adsorption, and yields elegant lyophilized cakes with acceptable clinically relevant reconstitution times. This research also demonstrates the benefit of utilizing specific vaccine formulation excipients and the effectiveness of excipient combinations that may be beneficial for other multivalent adjuvant containing vaccines to enable novel lyophilized formulations necessary for improved global vaccine access.

Quantitative Analysis of Vaccine Antigen Adsorption to Aluminum Adjuvant Using an Automated High-Throughput Method.

Aluminum-containing adjuvants have been widely used in vaccine formulations to safely and effectively potentiate the immune response. The examination of the extent of antigen adsorption to aluminum adjuvant is always evaluated during the development of aluminum adjuvant containing vaccines. A rapid, automated, high-throughput assay was developed to measure antigen adsorption in a 96-well plate format using a TECAN Freedom EVO® (TECAN). The antigen adsorption levels at a constant adjuvant concentration for each sample were accurately measured at 12 antigen/adjuvant (w/w) formulation ratios. These measurements were done at aluminum adjuvant concentrations similar to normal vaccine formulations, unlike previous non-automated and automated adjuvant adsorption studies. Two high-sensitivity analytical methods were used to detect the non-absorbed antigens. The antigen-to-adjuvant adsorption curves were fit to a simple Langmuir adsorption model for quantitatively analyzing the antigen to the adjuvant adsorption level and strength. The interaction of two model antigens, bovine serum albumin and lysozyme, with three types of aluminum adjuvant, were quantitatively analyzed in this report. Automated, high-throughput methodologies combined with sensitive analytical methods are useful for accelerating practical vaccine formulation development.LAY ABSTRACT: Vaccines are probably the most effective public health method to prevent epidemics of many infectious diseases. Many of the most effective vaccines contain aluminum adjuvant. This report describes novel technology that can be used to better optimize the efficacy and stability of aluminum adjuvant-containing vaccines.

Accelerating Vaccine Formulation Development Using Design of Experiment Stability Studies.

Vaccine drug product thermal stability often depends on formulation input factors and how they interact. Scientific understanding and professional experience typically allows vaccine formulators to accurately predict the thermal stability output based on formulation input factors such as pH, ionic strength, and excipients. Thermal stability predictions, however, are not enough for regulators. Stability claims must be supported by experimental data. The Quality by Design approach of Design of Experiment (DoE) is well suited to describe formulation outputs such as thermal stability in terms of formulation input factors. A DoE approach particularly at elevated temperatures that induce accelerated degradation can provide empirical understanding of how vaccine formulation input factors and interactions affect vaccine stability output performance. This is possible even when clear scientific understanding of particular formulation stability mechanisms are lacking. A DoE approach was used in an accelerated 37(°)C stability study of an aluminum adjuvant Neisseria meningitidis serogroup B vaccine. Formulation stability differences were identified after only 15 days into the study. We believe this study demonstrates the power of combining DoE methodology with accelerated stress stability studies to accelerate and improve vaccine formulation development programs particularly during the preformulation stage.

Characterization of Propylene Glycol-Mitigated Freeze/Thaw Agglomeration of a Frozen Liquid nOMV Vaccine Formulation by Static Light Scattering and Micro-Flow Imaging.

UNLABELLED: The purpose of this work was to investigate the susceptibility of an aluminum adjuvant and an aluminum-adjuvanted native outer membrane vesicle (nOMV) vaccine formulation to freeze/thaw-induced agglomeration using static light scattering and micro-flow Imaging analysis; and to evaluate the use of propylene glycol as a vaccine formulation excipient by which freeze/thaw-induced agglomeration of a nOMV vaccine formulation could be mitigated. Our results indicate that including 7% v/v propylene glycol in an nOMV containing aluminum adjuvanted vaccine formulation, mitigates freeze/thaw-induced agglomeration. LAY ABSTRACT: We evaluated the effect of freeze-thawing on an aluminum adjuvant and an aluminum adjuvanted native outer membrane vesicle (nOMV) vaccine formulation. Specifically, we characterized the freeze/thaw-induced agglomeration through the use of static light scattering, micro-flow imaging, and cryo-electron microscopy analysis. Further, we evaluated the use of 0-9% v/v propylene glycol as an excipient which could be included in the formulation for the purpose of mitigating the agglomeration induced by freeze/thaw. The results indicate that using 7% v/v propylene glycol as a formulation excipient is effective at mitigating agglomeration of the nOMV vaccine formulation, otherwise induced by freeze-thawing.

Detecting and preventing reversion to toxicity for a formaldehyde-treated C. difficile toxin B mutant.

The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders of magnitude by a combination of targeted point mutations. However, a TcdB mutant with five point mutations (referred to herein as mTcdB) still has residual toxicity that can be detected in cell-based assays and in-vivo mouse toxicity assays. This residual toxicity can be effectively removed by treatment with formaldehyde in solution. Storage of the formaldehyde-treated mTcdB as a liquid can result in reversion over time back to the mTcdB level of toxicity, with the rate of reversion dependent on the storage temperature. We found that for both the "forward" mTcdB detoxification reaction with formaldehyde, and the "reverse" reversion to toxicity reaction, mouse toxicity correlated with several biochemical assays including anion exchange chromatography retention time and appearance on SDS-PAGE. Maintenance of a low concentration of formaldehyde prevents reversion to toxicity in liquid formulations. However, when samples with 0.016% (v/v) formaldehyde were lyophilized and stored at 37 °C, formaldehyde continued to react with and modify the mTcdB in the lyophilized state. Lyophilization alone effectively prevented reversion to toxicity for formaldehyde-treated, formaldehyde-removed mTcdB samples stored at 37 °C for 6 months. Formaldehyde-treated, formaldehyde-removed lyophilized mTcdB showed no evidence of reversion to toxicity, appeared stable by several assays, and was immunogenic in mice, even after storage for 6 months at 37 °C.

Inhibition of tungsten-induced protein aggregation by cetyl trimethyl ammonium bromide.

TECHNICAL ABSTRACT: The purpose of this work was to investigate a potential mechanism for the inhibition of tungsten-mediated monoclonal antibody (mAb) biophysical modifications and sub-visible particle formation. A 1 mg/mL mAb formulated in 150 mM NaCl, 20 mM histidine, pH 6.0, was incubated with 1, 37, and 100 ppm of tungsten polyanions in the form of sodium tungstate both in the presence and absence of the anionic surfactant and chelating agent diethylene triamine pentaacetic acid (DTPA) or the cationic surfactant cetyl trimethyl ammonium bromide (CTAB) for 24 h at 25 °C. Assays including pH, UV-Vis spectroscopy, size exclusion chromatography, intrinsic tryptophan/tyrosine fluorescence, and micro-flow imaging were performed to assess the impact on short-term mAb stability and aggregation. We conclude that the use of micromolar concentrations of the formulation excipient and cationic surfactant CTAB equivalent to the anticipated tungsten concentration in solution effectively inhibits loss of protein concentration, fragmentation, changes in intrinsic fluorescence intensity, and the formation of sub-visible particles. LAY ABSTRACT: The purpose of this work was to investigate a potential mechanism for the inhibition of tungsten-mediated monoclonal antibody (mAb) biophysical modifications and sub-visible particle formation. A mAb formulation was incubated with tungsten polyanions in the presence and absence of the anionic surfactant and chelating agent diethylene triamine pentaacetic acid (DTPA) or the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). Formulation was characterized by pH, UV-Vis spectroscopy, size exclusion chromatography, intrinsic tryptophan/tyrosine fluorescence, and micro-flow imaging. We conclude that the formulation excipient and cationic surfactant CTAB effectively inhibits biophysical modifications and sub-visible particle formation.

Influence of formulation pH and suspension state on freezing-induced agglomeration of aluminum adjuvants.

Freezing and thawing of vaccines containing aluminum adjuvants can lead to formation of aggregates and loss in vaccine potency. We sought to understand whether and to what extent the freeze-thaw damage to aluminum adjuvants would differ based on suspension state (flocculation and settlement) at the time of freezing. As flocculation and settlement characteristics of aluminum adjuvants are driven largely by the electrostatic charges on the adjuvant particles, which, in turn, are strongly influenced by the pH of the suspension, we conducted freeze-thaw studies on both Adjuphos and Alhydrogel™ samples at three pH levels (4, 6.5, and 7.2) in buffer solutions with 9% sucrose. Significantly less aggregation occurred in the buffered sucrose solutions at the pH furthest from the aluminum adjuvant point of zero charge during slow freezing at -20°C. The freezing-induced aggregation for the samples with 9% sucrose at each pH was minimal during fast freezing at -70°C and -115°C. Suspensions that were flocculated and settled to a greater extent experienced the most freeze-thaw aggregation, whereas suspensions that were frozen before significant flocculation and settlement occurred showed little or no aggregation. Because pH of formulation can affect flocculation and settling time, it indirectly affects the extent of freeze-thaw aggregation.