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.

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.

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

The surface stability and resulting transmission of severe acute respiratory syndrome coronavirus 2 (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 behavior 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, we first give 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.