VIRUSES

TRULY QUANTITATIVE MEASUREMENT OF TOTAL TITER AND SUBGROUP TITER OF VIRUS

Author: Nanofcm     Date: November 26, 2021

Virus is a general term for a group of tiny organisms that are made up of non-cellular forms of nucleic acid molecules (DNA or RNA) and proteins and live in parasitic life. Although viruses are small in size (20-200 nm), they are by far the most abundant ‘lifeforms’ in nature, and also the chief culprit of many fatal diseases. The pandemic caused by COVID-19 (SARS-CoV-2) has led to dramatic loss of human lives globally, and presents an unprecedented challenge to public health. An interpretation of the biological structure and function of novel Coronavirus and an efficacious vaccine are essential to prevent further morbidity and mortality. On the other hand, viruses have also been used more positively in the medical field. Virus particles have many natural characteristics that can be applied to nanobiology and nanomedicine, and there are unprecedented opportunities for related applications based on the properties of virus nanoparticles. Oncolytic viruses, a typical branch of new immuno-therapeutic agents, rely on both the selective tumor cell killing and the induction of systematic antitumor immunity to achieve antitumor responses, has shown great promise in tumor treatment, especially when combing with other therapies like immune checkpoint inhibitors. As an advanced targeted cancer therapy, chimeric antigen receptor (CAR) T cell therapy has unexpectedly fueled an increasing number of clinical trials for cancer treatment, and encouraging progress has been made continuously. Lentivirus has been extensively used as the gene delivery vector to transfer CAR gene to T cells. Moreover, adeno associated viruses (AAV) are also common vectors for gene transfer in vivo, which show great potential in therapeutic applications. Lentivirus and AAV are two most frequently used vectors for cell and gene therapy and have a lot of advantages such as excellent safety, low immunogenicity, able to infect both mitotic and non-mitotic cells, and able to mediate long-term stable gene expression. With the development of scientific research, rapid detection and accurate description of viruses have become more and more important, which is essential for the applications. The physical and chemical properties of viruses, such as size, concentration, biochemical component as well as the packaging efficiency of functional molecules would cause direct effect to their applications. Transmission electron microscopy (TEM) is still the most recognized means to measure the size and morphology of a virus despite the fact that it is labor intensive and time-consuming. Conventional plaque titer and TCID50 method are the most classical approaches for concentration measurement of infectious viruses, however, they quantify only those which cause visible cell-damage thus exclude the viruses without infectivity. Therefore, a virus analysis method which can quickly and reliably detect, quantify and characterize virus particles with single particle sensitivity is highly needed. Here, the Flow NanoAnalyzer is introduced, which can directly detect MS2 virus with particle size as low as 27 nm by light scattering. The NanoFCM platform provides a versatile and powerful platform for the multiparameter analysis of individual viruses.

1. Quality Control of COVID-19 Vaccine

Quality control is indispensable to ensure the adequate purity of virus products in the biopharmaceutical industry. Purity assessment of virus products is indispensable in many biotechnology applications, especially in manufacturing vaccines. In the process of preparing virus products, many particles can coexist in the lysates of the host cells, including cell debris, empty capsids, and mature virions. What’s more, long-term tracking of the stabiilty is essential to maintain the high efficacy. Here a COVID-19 vaccine made from inactived Coronavirus is analyzed. The size (diameter) distribution and particle concentration of single novel Coronavirus could be acquired directly from the software of Flow NanoAnalyzer. The uniformity of viral particles is evaluatd directly, while purity can be assessed by nucleic acid staining. The purity of COVID-19 increased to nearly 100% after several rounds of purification steps. The Flow NanoAnalyzer platform enables quantitative and multiparameter analysis of single Coronavirus, which is distinctively sensitive, yet high-throughput, and shows great potential in quality control of COVID-19 vaccine.

2.Comprehensive Analysis of Lentivirus for Cell and Gene Therapy

Chimeric antigen receptor (CAR) T cell Therapy for B cell malignancies is the first cell therapy approved by the US Food and Drug Administration for the treatment of cancer. After that, an increasing number of clinical trials are being conducted on CAR-T cell therapy. Lentivirus is the most widely used vector in CAR-T cell therapy. A complete CAR-T cell process includes the preparation of lentiviral vectors and the production of CAR-T cell products. Comprehensive characterization of lentiviruses is crucial to the successful construction of CAR T.

The size distribution of lentivirus is determined through direct light scattering detection. Membrane permeable nucleic acid dye is used to label the RNA, while the VSV-G protein is specifically recognized by fluorescent-tagged monoclonal antibody. A particle with both RNA signal and VSV-G signal is identified as an effective lentivirus vector. Dual-label analysis of nucleic acid and protein of lentiviral vectors allows not only to optimize the culturing conditions and to maximize the production of the virus, but also to screen the virus strains that effectively express the capsule proteins.

In addition to the direct measurement of lentivirus concentration and purity, the Flow NanoAnalyzer can be applied to all steps of the lentivirus manufacturing, from R&D, production, purification and quality control, to facilitate the large-scale production of lentivirus, and to speed up the promotion and application of CAR-T therapy.

3. Oncolytic Adenovirus for Treatment of Tumor

Oncolytic Viruses are a type of tumor-killing virus with replication ability, which selectively infect tumor cells by deactivating tumor suppressor genes in target cells, replicating themselves in their cytoplasm and eventually destroying them. It also stimulates the immune response, attracting more immune cells to continue killing residual cancer cells. Newcastle disease virus, herpes simplex virus, reovirus and adenovirus have all been modified into oncolytic viruses due to their oncotropic properties, which have the function of specific recognition and lysis of tumor cells, while have no killing effect on normal somatic cells. Oncolytic Adenoviruses (OA) have been explored in both preclinical and clinical therapies. While the poor targeting delivery is a major obstacle that limits OA application. Here OA was decorated with bioengineered cell membrane nanovesicles (BCMNs) that are genetically engineered with targeting ligands, the resulting OA@BCMNs exhibited enhanced targeting delivery, vial oncolysis, and survial benefits in multiple xenograft models. Nano-flow cytometry was used to evaluate the encapsulation efficiency of OA@BCMNs, CFSE and SYTO 62 probes were applied to label the BCMNs and nucleic acids of OA, respectively. The positive ratio of 60% demonstrate the successful preparation of the OA-encapsulated BCMNs nanostructure.

4. Virus-based drug delivery

Virus particles can be regarded as natural nucleic acid nanomaterials, which consist of nucleic acid (drugs) wrapped in an empty virus shell (drug carrier). Through nucleic acid staining, the complete virus, empty virus shell, free nucleic acid and other components can be quickly distinguished from the mixture, and further reveal the drug packaging efficiency, the amount of package, and the proportion of effective drugs and other parameters.

5. Viral Vaccines

The production process of viral vaccine includes virus culture, harvest, purification and emulsification. By nucleic acid staining, the Flow NanoAnalyzer can quickly assess the virus at different stages of production, providing information on particle concentration and purity. According to the experimental results,with the further purification process, the concentration of virus particles gradually decreased, but the purity showed an upward trend.

6. Adeno-associated Viruses

Adeno-associated viruses (AAV)(diameter: 20-25 nm)are common gene delivery vectors in vivo, which show potential in therapeutic applications. Encouraging results of AAV vectors have been observed in preclinical models. To be specific, the delivery of CRISPR/Cas9 nucleases via AAV vectors displays therapeutic utility in preclinical models of a variety of diseases, and this approach is being rapidly applied to clinical trials. The quality control/evaluation of encapsulation ratio has been the speed-limiting process for the development of AAV based therapies. The particle concentration of AAV can be measured by non-specific labeling of capsid proteins, while the mature viruses with nucleic acid is identified through nucleic acid staining, the encapsulation ratio is easily provided.

7. Purity Assessment of Adenovirues

Through side scattering detection and concurrent fluorescence detection by nucleic acid staining, purity analysis of adenoviruses is quickly achieved.

● High Sensitivity ——Label-Free Detection of Single Viruses

The Flow NanoAnalyzer allows you to quickly detect virus nanoparticles in a single particle without labeling. The virus used in this experiment is levivirus MS2, which is one kind of non-enveloped, spherical virion about 27 nm in diameter, and the genome is monopartite, linear ssRNA (+) about 3.5 kb in size. The signal-to-noise (S/N) ratio, which is calculated as the average burst height of all the nanoparticles detected in 1 min divided by the standard deviation of the background signal (noise), is 11 for the MS2 viruses, indicating that the Flow NanoAnalyzer provides exceptional sensitivity in discerning MS2 viruses against the background noise. This sensitivity could meet the detection demand of most virus nanoparticles in nature.

● Exceptional Resolution And High Throughput ——Discrimination and Size Measurement of Viruses in a Mixture

In addition to single particle characterization of viruses, the Flow NanoAnalyzer can also perform discrimination and size measurement of viruses in a mixture. A standard calibration curve is constructed with monodisperse silica nanospheres of five different diameters ranging from 43-113 nm. The SS burst areas of silica nanoparticles are plotted as a function of the diameters determined by TEM, and the SS burst area of every single virus is converted to the corresponding particle size. Employing silica nanoparticles with five different sizes to calibrate a standard curve, rapid and accurate virus size measurements with a resolution comparable to that of TEM is achieved. Particularly, with a throughput up to 10,000 particles per minute, a statistically reliable size distribution profile can be obtained in 2-3 minutes.

● Purity Assessment and Dynamic Monitoring of the Viral Genome Release Process

Quality control is indispensable to ensure the adequate purity of virus products in the biopharmaceutical industry. Purification assessment of virus products is indispensable in many biotechnology applications. In the process of preparing virus products, many particles can coexist in the lysates of the host cells infected with a wild type phage, including cell debris, DNA-free proheads, empty capsids (after DNA ejection), and mature virions. In the meanwhile, the natural process of viruses delivering their genes into the hosts for self-replication has inspired the design of virus-like particles to deliver therapeutic or imaging agents. Monitoring the viral genome release process will allow for deeper insights into the mechanisms of the cargo release.  In this study, T7 phage was treated with NaClO4 to induce its genome release. The Flow NanoAnalyzer be used to analyze the composition of the viral sample and to monitor the viral genome release process following treatment with NaClO 4 .

On the other hand, viruses bear exceptional stability and biocompatibility, and the particles present programmable units, which can be modified by either genetic modification or chemical biological conjugation methods. The high transfection efficiency of viral gene-therapy vectors and their monodisperse structures with precise shapes and sizes also make viral particles powerful drug-delivery vehicles and versatile nanotechnology building blocks. Further control of the self-assembly process in host cells can realize the modification of virus particles, so as to develop beneficial applications for human beings,such as medical diagnosis and treatment of reagents, gene delivery carriers and antimicrobial agents. High sensitivity, exceptional resolution and high throughput characteristics of this instrument made it an effective means for single particle multi-parameter characterization of viral particles. The measurement of viruses will benefit from the concurrent detection of side scatter and fluorescence of Flow NanoAnalyzer, which allows the quantification of both mature viruses and empty capsids. Furthermore, this method allows us to estimate the concentration of non-infectious virus-like particles, which provides a rapid and simple platform for the development of viral nanotechnology.