Single-particle chemical force microscopy to characterize virus surface chemistry

Two important viral surface characteristics are the hydrophobicity and surface charge, which determine the viral colloidal behavior and mobility. Chemical force microscopy allows the detection of viral surface chemistry in liquid samples with small amounts of virus sample. This single-particle method requires the functionalization of an atomic force microscope (AFM) probe and covalent bonding of viruses to a surface. A hydrophobic methyl-modified AFM probe was used to study the viral surface hydrophobicity, and an AFM probe terminated with either negatively charged carboxyl acid or positively charged quaternary amine was used to study the viral surface charge. With an understanding of viral surface properties, the way in which viruses interact with the environment can be better predicted

Surface Chemistry Can Unlock Drivers of Surface Stability of SARS-CoV-2 in Variety of Environmental Conditions

The surface stability and resulting transmission of the SARS-CoV-2, specifically in indoor environments, have been identified as a potential pandemic challenge requiring investigation. This novel virus can be found on various surfaces in contaminated sites such as clinical places, however, the behaviour and molecular interactions of the virus with respect to the surfaces are poorly understood. Regarding this, the virus adsorption onto solid surfaces can play a critical role in transmission and survival in various environments. In this article, firstly an overview of existing knowledge concerning viral spread, molecular structure of SARS-CoV-2, and the virus surface stability is presented. Then, we highlight potential drivers of the SARS-CoV-2 surface adsorption and stability in various environmental conditions. This theoretical analysis shows that different surface and environmental conditions including temperature, humidity, and pH are crucial considerations in building fundamental understanding of the virus transmission and thereby improving safety practices

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

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

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

Scalable method utilizing low pH for DNA removal in the harvest of recombinant adeno-associated virus vectors

Adeno-associated virus (AAV) is a strong candidate for single-gene mutation gene therapy. AAV comes in several serotypes that target different organs in the body. The current purification methods for AAV vectors often rely on serotype dependent affinity chromatography. However, it is desired to create a platform for AAV purification that mirrors the evolution of antibody platform processes. To do this, any serotype dependent steps need to be removed from the process. The harvest and initial capture steps that can satisfy all of the needs of a platform AAV process is the use of low pH and Triton in the harvest, followed by filtration and cation exchange chromatography (CEX) for initial capture. The low pH hydrolyses and removes the host cell DNA, a difficult contaminate to remove. CEX then provides a concentration and capture step. The only step that remains is to determine the polishing and final formulation. This harvest strategy provides a serotype independent purification that removes both host cell DNA and host cell proteins and is friendly to scale-up for future AAV processes

Adsorption of a non-enveloped mammalian virus to functionalized nanofibers

In the pursuit of finding superior methods to remove pathogens from drinking water, this study examines the adsorption of a non-enveloped, mammalian virus to highly charged nanofibers. N-[(2-Hydroxyl-3-trimethylammonium) propyl] chitosan (HTCC) nanofibers were synthesized by the addition of a quaternary amine to chitosan. HTCC was blended with polyvinyl alcohol (PVA) to produce nanofibers by electrospinning. The nanofibers were stabilized against water by crosslinking with glutaraldehyde. When studied in the range of 100-200. nm in diameter, larger fibers were able to adsorb about 90% more virus than smaller fibers. The kinetics of the adsorption was modeled with pseudo-first order kinetics and equilibrium was achieved in as little as 10. min. Equilibrium adsorption was modeled with the Freundlich isotherm with a Freundlich constant of 1.4. When the Freundlich constant deviates from 1, this demonstrates that there is heterogeneity at the adsorption surface. The heterogeneity likely occurs at the nanofiber surface since a polymeric blend of two polymers was used to electrospin the nanofibers. The model mammalian virus, porcine parvovirus (PPV), has a fairly homogeneous, icosahedral protein capsid available for adsorption. The fast adsorption kinetics and high capacity of the nanofibers make HTCC/PVA a potential filter material for the removal of pathogens from drinking water. © 2014 Elsevier B.V

Challenges in downstream purification of gene therapy viral vectors

The diversity and application of viral products has continued to explode. These modalities can be vaccines, cancer therapies, and gene therapies. However, their diversity is also the challenge in the downstream processing. Viral particles can be very labile, thus requiring intimate knowledge of biology to create environments where they are stable. However, despite these challenges, we have created many processes that produce large amounts of viral products. Different purification methods are utilized throughout the process. New modalities of chromatography are overcoming many of the challenges of diffusion-limited beads. Of special concern for gene therapy vectors is the need to separate the empty capsids from the full capsids, which contain the therapeutic gene of interest. With the discovery of novel therapuetic modalities that could revolutionize care by finding cures, the downstream processing of viral therapies needs to find solutions to make these therapies and cures affordable

Enveloped virus flocculation and removal in osmolyte solutions

© 2015 Elsevier B.V. Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a \u3e 80% removal with a 0.2. μm microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3. M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, this method has the potential to be a future platform purification process for vaccines