Virus Quantitation & Analysis using qViro
qViro uses size tunable nanopores combined with resistive pulse sensing to provide previously unavailable capability for rapid quantitation and analysis of viruses and viral aggregates in liquid suspension.
The size, concentration and relative surface charge of virus particles can be rapidly and accurately determined with single particle resolution. Analysis of thousands of viral particles usually takes less than 15 minutes. Key applications include characterization of viral particles for vaccine development and accurate analysis of vectors for gene therapy research
Table of Contents
Comparison with Infectivity Assays
Advantages and Disadvantages of Existing Methods
Example: qViro Analysis of Adenovirus
Example: qViro Analysis of Lentivirus
Vaccine Development
Vaccine development requires stable preparations with information on the number of virus particles present and how many of them are capable of infecting cells. Infectivity assays can answer the latter question but accurate determination of total virus numbers is difficult. Izon's qViro analyzer, which determines concentration (in particles/mL) by applying the principle of resistive pulse sensing, can be used to accurately correlate the number of virus particles to virus infectious units (IFU) when combined with standard titer analysis methods. The ability to accurately and reliably measure viral aggregation, is a further benefit of the system not available through alternative methods.
Comparison with Infectivity Assays
qViro provides the ability to rapidly and reliably size and count the total number of virus particles present in a sample. Used in conjunction with infectivity assays this allows users to determine the relative concentrations of infective virus particles vs. total viral particles present.
As inactivated ("killed") viral vaccines are no longer infectious they can not be assessed by infectivity assays, which require replicating and infectious virus for assessment via measurement of cytopathic effects or plaque forming units.
In the case of live attenuated vaccines, virus particles which have lost infectivity during the production process can not be detected by traditional methods. It is well known that the relative contribution of the non-infective component can vary considerably during manufacturing. Being able to accurately measure this component with qViro has significant implications for optimizing product yield during the manufacturing process.
Furthermore, as non-infective units may also induce an immune response when administered to patients, an understanding of their relative contribution to the final product is important.
The table below summarizes some of the main differences between qViro and biological virus quantitation methods (TCID50 and Plaque Assays).
Another advantage of the qViro system is the provision of accurate size distribution output, useful for assessment of viral aggregation and stability during vaccine production.
As particle-by-particle sizing occurs, the total number of viral aggregates can be measured and displayed in relation to the total number of pure virus in the sample. Measurement of Adenoviral and Lentiviral samples below clearly shows resolution of distinct aggregation peaks - corresponding to doublet and triplet formation.
Gene Therapy Research
The primary concern of gene therapy research is the safety and effectiveness of the viral vectors used. Regulatory agencies demand a wide range of tests on the safety and efficacy of gene therapy after the development of usable vectors and before approving clinical studies. There is a need to develop supportive technologies for gene transfer beyond actual vector research such as measurement of total viral particle concentration and viable viral titre.
The qViro provides previously unavailable capability to count and characterize individual virus particles in real-time. Applications include measurement of common gene therapy vectors including viral (e.g., adenovirus, lentivirus, granulovirus) and non-viral vectors (e.g., CaP/DNA/PEI hybrid particles, exosomes).
Advantages and Disadvantages of Existing Methods
Existing methods for evaluating quality and quantity of virus particles in biological solutions are limited and their pros and cons are listed below:
- Absorbance-based assays are commonly used especially because of their speed and simplicity; however the data obtained is highly variable and not accurate enough to determine precise viral count.
- Immuno assays are more accurate but at the cost of longer time and reagent consuming procedures.
- P24 assays are highly expensive but has become the de-facto lab standard. The requirement of clean room facilities, several hours of bench time and highly skilled operators can be considered a disadvantage
- P24 lentivirus assay is highly expensive, takes two to five days and the result does not provide the accurate viral particle number.
For non-viral gene therapy vectors, there are no reliable methods available for accurate, repeatable and quick particle titre determination. Current viral titration methods are shown in Table 1.
Table 1. Current viral titration methods (for adenovirus and lentivirus)
(Source: Adapted from Dr. Lijun Wang, Oxford University, Unpublished report)
|
Assay type |
Require transduction |
Assay principle |
Information |
Time |
Price per test |
|
Rapid relative |
no |
Nuclei Acid content |
Total viral particles |
30-60 min |
Low cost |
|
End point dilution |
yes |
Immureorganisation |
Viable virus (adenovirus) Relative(lenti) |
2-5 days |
~$100 for adenovirus ~$500 for lentivirus |
|
qPCR |
yes |
qPCR for viral DNA/RNA |
Relative conc. Viable virus |
Transduction 2-5 days, qPCR, 4hours. |
~$100 |
|
Marker gene detection |
yes |
FACS |
Viable virus |
Transduction 2-5days, FACS 30 min |
Expensive |
|
qViro |
no |
Nanopore |
Total viruses |
30min |
$5 to $55* |
Example: qViro Analysis of Adenovirus
Most frequently used vectors in gene therapy are the Adenoviral vectors and the first gene therapy product (Gendicine) that obtained license is an adenovirus to treat cancer.
The results produced for adenovirus after measurement on the qNano are shown as a standard size distribution histogram in Figure 1. The vertical axis shows the number of counts after counting on a particle-by-particle basis and the horizontal axis shows the virus diameter in nanometers, displaying the exact size distribution of the adenoviral population. This size distribution shows that most of the sample is in the 90-100nm range and a second aggregation peak is seen at 110nm virus diameter
Figure 1. Size Distribution Histogram for Adenovirus using qViro.
Example: qViro Analysis of Lentivirus
Lentivirus has been increasingly used as a gene therapy vector, however, until now there has been no available quantification technique for measuring it. The qViro instrument has been used to measure the size and concentration in particles per milliliter for this vector. Measurement of the lentivirus confirmed that the virus particles have a negative surface charge, as the particles moved towards a positive electrode under electrophoresis. The size distribution revealed polydispersity in the size of particle including visible dimer and trimer aggregate peaks showing some aggregation between viral particles as shown in Figure 2.
Figure 2. Size distribution of lentivirus after qViro analysis shows aggregation effects.
Lentivirus Concentration Analysis
Concentration values (particles*mL-1) were measured for lentivirus samples. Standard measurement values at a single applied pressure setting are used as the reference against which other samples are compared to obtain a precise absolute concentration value. By this method the viral particle concentration was measured as 4.7x1011 particles mL-1. This procedure is accurate to within 10% of the true concentration value excluding error in the reference calibration standard.
Conclusion
The qViro has been proven as a quick, robust method to quantify virus particles in terms of both size distribution and determination of physical titre. The compact size allows measurement inside a biological safety cabinet and experiments can be conducted during normal laboratory procedures. Due to the existing technology limitations, there is increased demand for developing better methods of virus quantification. qViro provides a noteworthy enhancement over alternative virus quantification techniques.
For more information please contact us at info@izon.com
