Design Rules for Sequestration of Viruses into Polypeptide Complex Coacervates

Encapsulation is a strategy that has been used to facilitate the delivery and increase the stability of proteins and viruses. Here, we investigate the encapsulation of viruses via complex coacervation, which is a liquid–liquid phase separation resulting from the complexation of oppositely charged polymers. In particular, we utilized polypeptide-based coacervates and explored the effects of peptide chemistry, chain length, charge patterning, and hydrophobicity to better understand the effects of the coacervating polypeptides on virus incorporation. Our study utilized two nonenveloped viruses, porcine parvovirus (PPV) and human rhinovirus (HRV). PPV has a higher charge density than HRV, and they both appear to be relatively hydrophobic. These viruses were compared to characterize how the charge, hydrophobicity, and patterning of chemistry on the surface of the virus capsid affects encapsulation. Consistent with the electrostatic nature of complex coacervation, our results suggest that electrostatic effects associated with the net charge of both the virus and polypeptide dominated the potential for incorporating the virus into a coacervate, with clustering of charges also playing a significant role. Additionally, the hydrophobicity of a virus appears to determine the degree to which increasing the hydrophobicity of the coacervating peptides can enhance virus uptake. Nonintuitive trends in uptake were observed with regard to both charge patterning and polypeptide chain length, with these parameters having a significant effect on the range of coacervate compositions over which virus incorporation was observed. These results provide insights into biophysical mechanisms, where sequence effects can control the uptake of proteins or viruses into biological condensates and provide insights for use in formulation strategies

Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction

The subset of plasma extracellular vesicles (EVs) that coprecipitate with low-density lipoprotein (LDL-EVs) carry coagulation and fibrinolysis pathway proteins as cargo. We investigated the association between LDL-EV hemostatic/fibrinolysis protein ratios and post-acute myocardial infarction (post-AMI) left ventricular (LV) remodeling which precedes heart failure. Protein concentrations of von Willebrand factor (VWF), SerpinC1 and plasminogen were determined in LDL-EVs extracted from plasma samples obtained at baseline (within 72 h post-AMI), 1 month and 6 months post-AMI from 198 patients. Patients were categorized as exhibiting adverse (n = 98) or reverse (n = 100) LV remodeling based on changes in LV end-systolic volume (increased or decreased ≥15) over a 6-month period. Multiple level longitudinal data analysis with structural equation (ML-SEM) model was used to assess predictive value for LV remodeling independent of baseline differences. At baseline, protein levels of VWF, SerpinC1 and plasminogen in LDL-EVs did not differ between patients with adverse versus reverse LV remodeling. At 1 month post-AMI, protein levels of VWF and SerpinC1 decreased whilst plasminogen increased in patients with adverse LV remodeling. In contrast, VWF and plasminogen decreased whilst SerpinC1 remained unchanged in patients with reverse LV remodeling. Overall, compared with patients with adverse LV remodeling, higher levels of SerpinC1 and VWF but lower levels of plasminogen resulted in higher ratios of VWF:Plasminogen and SerpinC1:Plasminogen at both 1 month and 6 months post-AMI in patients with reverse LV remodeling. More importantly, ratios VWF:Plasminogen (AUC = 0.674) and SerpinC1:Plasminogen (AUC = 0.712) displayed markedly better prognostic power than NT-proBNP (AUC = 0.384), troponin-I (AUC = 0.467) or troponin-T (AUC = 0.389) (p \u3c 0.001) to predict reverse LV remodeling post-AMI. Temporal changes in the ratios of coagulation to fibrinolysis pathway proteins in LDL-EVs outperform current standard plasma biomarkers in predicting post-AMI reverse LV remodeling. Our findings may provide clinical cues to uncover the cellular mechanisms underpinning post-AMI reverse LV remodeling

Multimodal peptide ligand extracts parvovirus from interface in affinity aqueous two-phase system

Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10 kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface

Prophylaxis and Remediation for Future Pandemic Pathogens—(Lessons from a Post-COVID World)

Since influenza and coronaviruses are currently deadly and emerging threats worldwide, better treatment, remediation and prevention options are needed. In that regard, a basic understanding of severe acute respiratory syndrome (SARS)-CoV-2/COVID-19 (Betacoronaviridae) and other viral pathogen mechanisms of transmission are expected. Unfortunately, unprecedented, and growing distrust of vaccines and even masks or personal protective equipment (PPE) in the United States and elsewhere presents itself as an added challenge. We postulate that development of improved and highly effective prophylactic measures, together with new life-saving therapies that do inhibit or otherwise treat infection of SARS-CoV-2, influenza and other viral pathogens, could be an adjunct measure to globally protect vulnerable individuals from pandemic threats. In this review, we share what we learned from the past COVID experience to offer a multifactorial and improved approach to current and future pandemic infections or threats using low-cost means

Empty and Full AAV Capsid Charge and Hydrophobicity Differences Measured with Single-Particle AFM

Adeno-associated virus (AAV) is showing promise as a therapy for diseases that contain a single-gene deletion or mutation. One major scale-up challenge is the removal of empty or non-gene of interest containing AAV capsids. Analytically, the empty capsids can be separated from full capsids using anion exchange chromatography. However, when scaled up to manufacturing, the minute changes in conductivity are difficult to consistently obtain. To better understand the differences in the empty and full AAV capsids, we have developed a single-particle atomic force microscopy (AFM) method to measure the differences in the charge and hydrophobicity of AAV capsids at the single-particle level. The atomic force microscope tip was functionalized with either a charged or a hydrophobic molecule, and the adhesion force between the functionalized atomic force microscope tip and the virus was measured. We measured a change in the charge and hydrophobicity between empty and full AAV2 and AAV8 capsids. The charge and hydrophobicity differences between AAV2 and AAV8 are related to the distribution of charge on the surface and not the total charge. We propose that the presence of nucleic acids inside the capsid causes minor but measurable changes in the capsid structure that lead to measurable surface changes in charge and hydrophobicity