Design Rules for the 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.

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.

Osmolyte enhanced aqueous two-phase system for virus purification.

Due to the high variation in viral surface properties, a platform method for virus purification is still lacking. A potential alternative to the high-cost conventional methods is aqueous two-phase systems (ATPSs). However, optimizing virus purification in ATPS requires a large experimental design space, and the optimized systems are generally found to operate at high ATPS component concentrations. The high concentrations capitalize on hydrophobic and electrostatic interactions to obtain high viral particle yields. This study investigated using osmolytes as driving force enhancers to reduce the high concentration of ATPS components while maintaining high yields. The partitioning behavior of porcine parvovirus (PPV), a nonenveloped mammalian virus, and human immunodeficiency virus-like particle (HIV-VLP), a yeast-expressed enveloped VLP, were studied in a polyethylene glycol (PEG) 12 kDa-citrate system. The partitioning of the virus modalities was enhanced by osmoprotectants glycine and betaine, while trimethylamine N-oxide was ineffective for PPV. The increased partitioning to the PEG-rich phase pertained only to viruses, resulting in high virus purification. Recoveries were 100% for infectious PPV and 92% for the HIV-VLP, with high removal of the contaminant proteins and more than 60% DNA removal when glycine was added. The osmolyte-induced ATPS demonstrated a versatile method for virus purification, irrespective of the expression system.

Changes in Membrane Dielectric Properties of Porcine Kidney Cells Provide Insight into the Antiviral Activity of Glycine.

The ability to monitor the status and progression of viral infections is important for development and screening of new antiviral drugs. Previous research illustrated that the osmolyte glycine (Gly) reduced porcine parvovirus (PPV) infection in porcine kidney (PK-13) cells by stabilizing the capsid protein and preventing virus capsid assembly into viable virus particles. Dielectrophoresis (DEP) was examined herein as a noninvasive, electric field- and frequency-dependent tool for real-time monitoring of PK-13 cell responses to obtain information about membrane barrier functionality and polarization. DEP responses of PK-13 cells were compared to those of PPV-infected cells in the absence and presence of the osmolyte glycine. With infection progression, PK-13 DEP spectra shifted toward lower frequencies, reducing crossover frequencies (fCO). The spherical single-shell model was used to extract PK-13 cell dielectric properties. Upon PPV infection, specific membrane capacitance increased over the time progression of virus attachment, penetration, and capsid protein production and assembly. Following glycine treatment, the DEP spectra displayed attenuated fCO and specific membrane capacitance values shifted back toward uninfected PK-13 cell values. These results suggest that DEP can be used to noninvasively monitor the viral infection cycle and screen antiviral compounds. DEP can augment traditional tools by elucidating membrane polarization changes related to drug mechanisms that interrupt the virus infection cycle.

Virus Isoelectric Point Determination Using Single-Particle Chemical Force Microscopy.

Virus colloidal behavior is governed by the interaction of the viral surface and the surrounding environment. One method to characterize the virus surface charge is the isoelectric point (pI). Traditional determination of virus pI has focused on the bulk characterization of a viral solution. However, virus capsids are extremely heterogeneous, and a single-particle method may give more information on the range of surface charge observed across a population. One method to measure the virus pI is chemical force microscopy (CFM). CFM is a single-particle technique that measures the adhesion force of a functionalized atomic force microscope (AFM) probe and, in this case, a virus covalently bound to a surface. Non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV) were used to demonstrate the use of CFM for viral particles with different surface properties. We have validated the CFM to determine the pI of PPV to be 4.8-5.1, which has a known pI value of 5.0 in the literature, and to predict the unknown pI of BVDV to be 4.3-4.5. Bulk measurements, ζ-potential, and aqueous two-phase system (ATPS) cross-partitioning methods were also used to validate the new CFM method for the virus pI. Most methods were in good agreement. CFM can detect the surface charge of viral capsids at a single-particle level and enable the comparison of surface charge between different types of viruses.

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.

Virus inactivation at moderately low pH varies with virus and buffer properties.

BACKGROUND: Virus inactivation is a critical operation in therapeutic protein manufacturing. Low pH buffers are a widely used strategy to ensure robust enveloped virus clearance. However, the choice of model virus can give varying results in viral clearance studies. Pseudorabies virus (SuHV) or herpes simplex virus-1 (HSV-1) are frequently chosen as model viruses to demonstrate the inactivation for the herpes family. RESULTS: In this study, SuHV, HSV-1, and equine arteritis virus (EAV) were used to compare the inactivation susceptibility at pH 4.0 and 4°C. SuHV and HSV-1 are from the same family, and EAV was chosen as a small, enveloped virus. Glycine, acetate, and citrate buffers at pH 4.0 and varying buffer strengths were studied. The inactivation susceptibility was found to be in the order of SuHV > HSV > EAV. The buffer effectiveness was found to be in the order of citrate > acetate > glycine. The smaller virus, EAV, remained stable and infectious in all the buffer types and compositions studied. CONCLUSION: The variation in inactivation susceptibility of herpes viruses indicated that SuHV and HSV cannot be interchangeably used as a virus model for inactivation studies. Smaller viruses might remain adventitiously infective at moderately low pH.

Single-Particle Characterization of SARS-CoV-2 Isoelectric Point and Comparison to Variants of Interest.

SARS-CoV-2, the cause of COVID-19, is a new, highly pathogenic coronavirus, which is the third coronavirus to emerge in the past 2 decades and the first to become a global pandemic. The virus has demonstrated itself to be extremely transmissible and deadly. Recent data suggest that a targeted approach is key to mitigating infectivity. Due to the proliferation of cataloged protein and nucleic acid sequences in databases, the function of the nucleic acid, and genetic encoded proteins, we make predictions by simply aligning sequences and exploring their homology. Thus, similar amino acid sequences in a protein usually confer similar biochemical function, even from distal or unrelated organisms. To understand viral transmission and adhesion, it is key to elucidate the structural, surface, and functional properties of each viral protein. This is typically first modeled in highly pathogenic species by exploring folding, hydrophobicity, and isoelectric point (IEP). Recent evidence from viral RNA sequence modeling and protein crystals have been inadequate, which prevent full understanding of the IEP and other viral properties of SARS-CoV-2. We have thus experimentally determined the IEP of SARS-CoV-2. Our findings suggest that for enveloped viruses, such as SARS-CoV-2, estimates of IEP by the amino acid sequence alone may be unreliable. We compared the experimental IEP of SARS-CoV-2 to variants of interest (VOIs) using their amino acid sequence, thus providing a qualitative comparison of the IEP of VOIs.

Physiochemical properties of enveloped viruses and arginine dictate inactivation.

BACKGROUND: Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. RESULTS: Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. CONCLUSIONS: Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane.

Thermostabilization of viruses via complex coacervation.

Widespread vaccine coverage for viral diseases could save the lives of millions of people each year. For viral vaccines to be effective, they must be transported and stored in a narrow temperature range of 2-8 °C. If temperatures are not maintained, the vaccine may lose its potency and would no longer be effective in fighting disease; this is called the cold storage problem. Finding a way to thermally stabilize a virus and end the need to transport and store vaccines at refrigeration temperatures will increase access to life-saving vaccines. We explore the use of polymer-rich complex coacervates to stabilize viruses. We have developed a method of encapsulating virus particles in liquid complex coacervates that relies on the electrostatic interaction of viruses with polypeptides. In particular, we tested the incorporation of two model viruses; a non-enveloped porcine parvovirus (PPV) and an enveloped bovine viral diarrhea virus (BVDV) into coacervates formed from poly(lysine) and poly(glutamate). We identified optimal conditions (i.e., the relative amount of the two polypeptides) for virus encapsulation, and trends in this composition matched differences in the isoelectric point of the two viruses. Furthermore, we were able to achieve a ∼103-104-fold concentration of virus into the coacervate phase, such that the level of virus remaining in the bulk solution approached our limit of detection. Lastly, we demonstrated a significant enhancement of the stability of non-enveloped PPV during an accelerated aging study at 60 °C over the course of a week. Our results suggest the potential for using coacervation to aid in the purification and formulation of both enveloped and non-enveloped viruses, and that coacervate-based formulations could help limit the need for cold storage throughout the transportation and storage of vaccines based on non-enveloped viruses.

Tie line framework to optimize non-enveloped virus recovery in aqueous two-phase systems.

Viral particle purification is a challenge due to the complexity of the broth, the particle size, and the need to maintain virus activity. Aqueous two-phase systems (ATPSs) are a viable alternative for the currently used and expensive downstream processes. This work investigated the purification of two non-enveloped viruses, porcine parvovirus (PPV), and human rhinovirus (HRV) at various ATPS tie lines. A polyethylene glycol (PEG) 12 kDa-citrate system at pH 7 was used to study the behavior of the partitioning on three different thermodynamic tie line lengths (TLLs). It was experimentally determined that increasing the TLL, and therefore increasing the hydrophobic and electrostatic driving forces within the ATPS, facilitated higher virus recoveries in the PEG-rich phase. A maximum of 79% recovery of infectious PPV was found at TLL 36 w/w% and tie line (TL) ratio 0.1. Increased loading of PPV was studied to observe the change in the partitioning behavior and similar trends were observed for all the TLs. Most contaminants remained in the citrate-rich phase at all the chosen TLLs, demonstrating purification of the virus from protein contaminants. Moderate DNA removal was also measured. Net neutral charged HRV was studied to demonstrate the effects of driving forces on neutrally charged viruses. HRV recovery trends remained similar to PPV on each TLL studied, but the values were lower than PPV. Recovery of viral particles in the PEG-rich phase of the PEG-citrate system utilized the difference in the surface hydrophobicity between virus and proteins and showed a direct dependence on the surface charge of each studied virus. The preferential partitioning of the relatively hydrophobic viral particles in the PEG-rich phase supports the hypothesis that both hydrophobic and electrostatic forces govern the purification of viruses in ATPS.

Biomimetic recyclable microgels for on-demand generation of hydrogen peroxide and antipathogenic application.

Microgels that can generate antipathogenic levels of hydrogen peroxide (H2O2) through simple rehydration in solutions with physiological pH are described herein. H2O2 is a widely used disinfectant but the oxidant is hazardous to store and transport. Catechol, an adhesive moiety found in mussel adhesive proteins, was incorporated into microgels, which generated 1-5 mM of H2O2 for up to four days as catechol autoxidized. The sustained release of low concentrations of H2O2 was antimicrobial against both gram-positive (Staphylococcus epidermidis) and gram-negative (Escherichia coli) bacteria and antiviral against both non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV). The amount of released H2O2 is several orders of magnitude lower than H2O2 concentration previously reported for antipathogenic activity. Most notably, these microgels reduced the infectivity of the more biocide resistant non-envelope virus by 3 log reduction value (99.9% reduction in infectivity). By controlling the oxidation state of catechol, microgels can be repeatedly activated and deactivated for H2O2 generation. These microgels do not contain a reservoir for storing the reactive H2O2 and can potentially function as a lightweight and portable dried powder source for the disinfectant for a wide range of applications. STATEMENT OF SIGNIFICANCE: Researchers have designed bioadhesives and coatings using the adhesive moiety catechol to mimic the strong adhesion capability of mussel adhesive proteins. During catechol autoxidation, hydrogen peroxide (H2O2) is generated as a byproduct. Here, catechol was incorporated into microgels, which can generate millimolar levels of H2O2 by simply hydrating the microgels in a solution with physiological pH. The sustained release of H2O2 was both antimicrobial and antiviral, inactivating even the more biocide resistant non-enveloped virus. These microgels can be repeatedly activated and deactivated for H2O2 generation by incubating them in solutions with different pH. This simplicity and recyclability will enable this biomaterial to function as a lightweight and portable source for the disinfectant for a wide range of applications.

A generalized purification step for viral particles using mannitol flocculation.

Vaccine manufacturing has conventionally been performed by the developed world using traditional unit operations like filtration and chromatography. There is currently a shift in the manufacturing of vaccines to the less developed world, requiring unit operations that reduce costs, increase recovery, and are amenable to continuous manufacturing. This work demonstrates that mannitol can be used as a flocculant for an enveloped and nonenveloped virus and can purify the virus from protein contaminants after microfiltration. The recovery of the virus ranges from 58 to 96% depending on virus, the filter pore size, and the starting concentration of the virus. Protein removal of 80% was achieved for the small nonenveloped virus using a 0.1 µm filter because proteins were not flocculated with the virus and flowed through the filter. It is hypothesized that mannitol dehydrates the viral surface by controlling the water structure surrounding the virus. Without the ability to become compact, as occurs with proteins, the virus aggregates in the presence of osmolytes and proteins do not. Osmolyte flocculation is a scalable process using high flux microfilters. It has been applied to both an enveloped and nonenveloped virus, making this process friendly to a variety of vaccine and gene therapy products. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1027-1035, 2018.