Oncolytic Viruses Are Designed to Grow in the Tumour Niche.

<p>There are at least six key critical features of tumour cell growth that can be targeted by oncolytic viruses. These include changes in the expression of viral-host cell receptors, the antiviral response, nucleotide and protein synthesis, cell proliferation, and apoptosis. A number of engineered or selected oncolytic viruses exist that can exploit one or more of these malignant characteristics.</p

Tumour Evolution.

<p>A hypothetical pathway of tumour evolution from a normal cell to an advanced-stage cancer. Mutations in key regulatory genes lead to changes in cell physiology that favour tumour growth. Over time, these genetic defects accumulate to confer many of the known hallmarks of cancer <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003836#ppat.1003836-Hanahan2" target="_blank">[131]</a>. The sequence of these events and the timing represented here is only one example of how this might occur.</p

Viral Quantitative Capillary Electrophoresis for Counting and Quality Control of RNA Viruses

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze–thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10⁸ to 10¹³ ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation

Aptamer-Based Viability Impedimetric Sensor for Viruses

The development of aptamer-based viability impedimetric sensor for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers to intact vaccinia virus were selected using cell-SELEX technique and integrated into impedimetric sensors via self-assembly onto a gold microelectrode. Remarkably, this aptasensor is highly selective and can successfully detect viable vaccinia virus particles (down to 60 virions in a microliter) and distinguish them from nonviable viruses in a label-free electrochemical assay format. It also opens a new venue for the development of a variety of viability sensors for detection of many microorganisms and spores

Anti-Fab Aptamers for Shielding Virus from Neutralizing Antibodies

Oncolytic viruses are promising therapeutics that can selectively replicate in and kill tumor cells. However, repetitive administration of viruses provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effect. In this work, we selected DNA aptamers against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies to shield the virus from nAbs and enhance its in vivo survival. For the first time, we used flow cytometry and electrochemical immunosensing to identify aptamers targeting the Fab region of antibodies. We evaluated the aptamers in a cell-based infection assay and found that several aptamer clones provide more than 50% shielding of VSV from nAbs and thus have the potential to enhance the delivery of VSV without compromising the patient’s immune system. In addition, we developed a bifunctional label-free electrochemical immunosensor for the quantitation of aptamer-mediated degree of shielding and the amount of vesicular stomatitis virus (VSV) particles. Electrochemical impedance spectroscopy was employed to interrogate the level of VSV in a linear range from 5 × 10⁴ to 5 × 10⁶ PFU mL^–1 with a detection limit of 10⁴ PFU mL^–1

Electrochemical Sensing of Aptamer-Facilitated Virus Immunoshielding

Oncolytic viruses (OVs) are promising therapeutics that selectively replicate in and kill tumor cells. However, repetitive administration of OVs provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effects. In this work, we selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus (VSV), to protect it from nAbs. A label-free electrochemical aptasensor was used to evaluate the degree of protection (DoP). The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VSV-specific aptamer. Electrochemical impedance spectroscopy was employed to quantitate VSV in the range of 800–2200 PFU and a detection limit of 600 PFU. The aptasensor was also utilized for evaluating binding affinities between VSV and aptamer pools/clones. An electrochemical displacement assay was performed in the presence of nAbs and DoP values were calculated for several VSV-aptamer pools/clones. A parallel flow cytometric analysis confirmed the electrochemical results. Finally, four VSV-specific aptamer clones, ZMYK-20, ZMYK-22, ZMYK-23, and ZMYK-28, showed the highest protective properties with dissociation constants of 17, 8, 20, and 13 nM, respectively. Another four sequences, ZMYK-1, -21, -25, and -29, exhibited high affinities to VSV without protecting it from nAbs and can be further utilized in sandwich assays. Thus, ZMYK-22, -23, and -28 have the potential to allow efficient delivery of VSV through the bloodstream without compromising the patient’s immune system

Electrochemical Differentiation of Epitope-Specific Aptamers

DNA aptamers are promising immunoshielding agents that could protect oncolytic viruses (OVs) from neutralizing antibodies (nAbs) and increase the efficiency of cancer treatment. In the present Article, we introduce a novel technology for electrochemical differentiation of epitope-specific aptamers (eDEA) without selecting aptamers against individual antigenic determinants. For this purpose, we selected DNA aptamers that can bind noncovalently to an intact oncolytic virus, vaccinia virus (VACV), which can selectively replicate in and kill only tumor cells. The aptamers were integrated as a recognition element into a multifunctional electrochemical aptasensor. The developed aptasensor was used for the linear quantification of the virus in the range of 500–3000 virus particles with a detection limit of 330 virions. Also, the aptasensor was employed to compare the binding affinities of aptamers to VACV and to estimate the degree of protection of VACV using the anti-L1R neutralizing antibody in a displacement assay fashion. Three anti-VACV aptamer clones, vac2, vac4, and vac6, showed the best immunoprotection results and can be applied for enhanced delivery of VACV. Another two sequences, vac5 and vac46, exhibited high affinities to VACV without shielding it from nAb and can be further utilized in sandwich bioassays

Quantitative analysis of macropinosome size in fibroblasts co-expressing Rac1^V12 and DGKζ constructs.

<p>(A) Representative images of wild type MEFs transfected with myc-Rac1^V12 alone or cotransfected with the indicated HA-tagged DGKζ construct. Scale bars = 20 um. Magnified images of the boxed regions are shown at the right. Scale bars = 5 um. (B) Graph showing the average macropinosome size in wild type MEFs expressing Rac1^V12 alone (-) or Rac1^V12 and the indicated DGKζ constructs (wt, kd or M1). Values are the mean ± SEM from three independent experiments. Statistical analysis was performed by a one-way ANOVA followed by a Tukey post-hoc multiple comparison test. Asterisks denote a significant difference (p<0.05) between the indicated conditions. (C) Graph showing the cumulative frequency distribution of macropinosome size (um²). The inset shows the distribution of macropinosomes between 0 and 5 um².</p

Quantitative analysis of YFP-DGKζ Localization During Macropinocytosis.

<p>Ratiometric images of wild type and DGKζ-null cells expressing NMTD-mCherry and either YFP-DGKζ, or YFP alone, taken approximately 15–20 seconds after ruffle closure were analyzed as described in Materials and Methods. Individual pixel intensity ratios were calculated for regions immediately surrounding the macropinosomes. The intensity ratios for YFP/NMTD-mCherry (gray bars) and YFP-DGKζ/NMTD-mCherry (black bars) were sorted into bins and plotted as probability distributions. The data were modeled by a three-parameter log-normal distribution (red and blue lines, respectively). Pixel ratios greater than 1 indicate the protein is more concentrated than the mCherry-NMTD membrane marker, while values less than 1 indicate a lower concentration. Asterisks indicate a significant difference in the percentages of pixels in each bin with the indicated intensity ratio.</p

YFP-DGKζ Localization During Macropinosome Biogenesis.

<p>Wild type fibroblast cells were cotransfected with plasmids encoding the N-terminal membrane-targeting domain of neuromodulin fused to the N-terminus of mCherry (NMTD-mCherry) and either YFP-DGKζ or YFP alone. Cells stimulated with 50 ηg/ml PDGF were visualized by time-lapse video microscopy. Shown are representative images of YFP-DGKζ (A-E) and NMTD-mCherry (A’-E’) localization in a single optical plane from a time-lapse sequence during various stages of macropinosome biogenesis including: the formation of an irregular ruffle (A and B, horizontal arrows), transition to a curved ruffle (A, vertical arrow and C, horizontal arrow), closure into a circular ruffle or membrane cup (B and C, vertical arrows), and cup closure (D, vertical arrow and E, arrows). Note the high concentration of YFP-DGKζ surrounding the newly formed macropinosomes (D” and E”, vertical arrows). (F-F”) YFP alone also appears to be concentrated around newly formed macropinosomes. Scale bars = 10 um.</p