Aptamer-Facilitated Cryoprotection of Viruses

Abstract

Global vaccination and gene therapy programs have an urgent demand for stabilization of viral vectors at low temperature. We used a quadramer, a bridge-connected DNA tetra-aptamer to antivesicular stomatitis virus (VSV), as a viral cryoprotectant. Results showed that the tetravalent antivirus DNA aptamers protect viral activity during multiple freeze− thaw cycles, shield from neutralizing antibodies, and decrease aggregation of viral particles.KEYWORDS: Aptamer, quadramer, vesicular stomatitis virus, viral stabilizers, cryoprotection C ryoprotection of viral vectors is essential for the development of vaccines and anticancer therapeutics. Because of temperature sensitivity, many viruses should be stored frozen. 1 Therefore, delivery of active viruses depends on a cold chain, a distribution network set up to maintain optimal low temperatures during the transportation and storage. However, this cold chain system represents a major economic and logistical burden, estimated to be as high as 80% of the entire financial cost. 2 In addition, a viral vector should be infectious in the presence of a host's neutralizing antibodies (nAbs). In the work, we used vesicular stomatitis virus (VSV) as an important viral vector in targeted oncolytic immunotherapies. 3 It is an enveloped RNA rhabdovirus that consists of a lipid envelope enclosing a nucleocapsid and an associated matrix formed by M proteins. 4−6 VSV is innocuous and engineered to target malignant cells and also increase a tumor's susceptibility to chemotherapeutic agents and the host immune response. Furthermore, VSV is a suitable choice for vaccine vectors and also has a key role in immunity and virology research. One of the challenges of utilizing VSV is its limited stability under different handling and storage conditions. 7 Several agents have been employed to increase the low temperature stability of viruses as effective vaccines such as metal ions, albumin, and gelatin. 8−11 However, none of these compounds have provided satisfactory results for oncolytic viruses due to low cryoprotection potency or high cell toxicity. The stabilizing effect of glycoproteins on the VSV surface 12 inspired us to look at VSV-specific aptamers. The goal of this study was to determine whether aptamers can preserve the infectivity of VSV after multiple freeze−thaw cycles and in the presence of nAbs (Scheme 1).Results and Discussion. We used anti-VSV DNA aptamer clones Z23, Z29, S39, and M50 (Table 1) that were previously selected in our laboratory and have shown high affinity to VSV. 13 To increase avidity and serum stability, we designed a quadramer, which was made by connecting four aptamers (equimolar mixture of all above-mentioned aptamers), with an oligonucleotide bridge (Figure 1 and Scheme 1). We measured the apparent dissociation constant (K d ) for monomeric pool and quadramer using the approach described by Sefah et al. 14 The K d for monomeric pool of four clones was 71 ± 15 nM, and quadramer had affinity of 22 ± 9 nM. The protective role of the aptamers and q...

Anti-Proliferative Effect of Doxorubicin-Loaded AS1411 Aptamer on Colorectal Cancer Cell

“…Moreover, the aptamers rarely induce immune response. They have been applied in a number of applications including antivirus (Ghobadloo et al, 2014) anticancer (Ireson and Kelland, 2006) and drug delivery (Zhang et al, 2011). Previously, our study has demonstrated that using PDGF-BB aptamer to interfere with the binding of PDGF-BB to its receptor suppressed CRC cell proliferation in part via down-regulation of the Ras/Raf/MEK/ERK signaling pathway (Sae-Lim et al, 2019).…”

Forced Degradation Studies to Identify Critical Process Parameters for the Purification of Infectious Measles Virus

“…However, the Influenza A virus was much more susceptible, with one log reduction in infectivity after two cycles of freezing at –40 °C and thawing for 20 min at room temperature, and a seven log reduction after five cycles [34]. In the case of Vesicular stomatitis virus (VSV), infectivity declined by two log values after 10 freeze–thaw cycles but the precise conditions were not specified [35]. Therefore, it appears that the MV is more robust than the Influenza A virus and VSV with respect to the damage caused by freeze–thaw cycles, which is advantageous for the preparation of the virus particles.…”

Aptamers Enhance Oncolytic Viruses’ Antitumor Efficacy

“…Repeated administration of oncolytic viruses induces neutralizing antibodies (nAbs) production, decreasing the antitumor effect [ 97 ]. At the proof-of-concept level, novel methods for OVs shielding from nAbs have been proposed [ 49 , 50 , 98 ]. The antigen-binding fragment (Fab) region of the rabbit anti-vesicular stomatitis virus (VSV) neutralizing polyclonal antibodies was chosen as the target for aptamer selection.…”

“…In the works mentioned above [ 49 , 50 , 98 ], DNA-aptamer selection against VSVs was also carried out. It was shown that the combination of anti-VSV and anti-nAbs aptamers resulted in increased infectivity, indicating a synergistic mechanism.…”

“…These molecules can be used in media compositions to maintain vaccine quality and store virions and VLPs [ 108 ]. The quadramer of anti-VSV aptamers has also been shown to protect viral activity during multiple freeze–thaw cycles; in particular, increasing virus infectivity by 1.4 logs after 60 freeze–thaw cycles in a plaque formation assay prevents virus aggregation [ 98 ]. The latter is necessary for the further infection process because aggregation can limit the spread of viral particles and prevents the establishment of a successful infection in the early stages [ 109 ].…”