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Posted in | Nanomedicine | Nanoanalysis

Counting and Sizing Viruses Using NanoSight Instruments

Published on June 2, 2009 at 7:50 AM

The ability of the NanoSight instrument to count and size viruses and their aggregates in liquid suspension is becoming increasingly important to those involved in the development of viral vaccines.

Viral vaccines fall under 2 main categories:

  • Live (attenuated) vaccines
  • Killed (inactivated) vaccines

A live attenuated vaccine is one where the virulence of the virus has been reduced, such that when the vaccine is administered to the patient, it induces an immune response without causing clinical disease. The virus will replicate within the host and hence provide immunity for an extended period of time. The majority of successful viral vaccines fall under category including vaccines for measles, mumps, rubella, influenza, yellow fever and polio.

Inactivated viruses are used in cases where an appropriate attenuated vaccine has not been developed or in cases where the virus is thought to be likely to revert from the attenuated form, into a more virulent form of the virus. The virus cannot replicate within the body and hence there is typically a lower host response to the vaccine and often multiple doses are required. The most common inactivated viral vaccines include typhoid, rabies and polio.

NanoSight’s role in the virus purification process
The development of viral vaccines requires viruses to be cultured in live cells, harvested and then purified. Vaccine manufacturers are interested in monitoring the purity of the viral preparation at various key stages of the purification process and understand the concentration of virus material present. There is where the NanoSight instrument adds real value. The particle-by-particle approach to sizing and counting viruses can easily distinguish viruses from larger cell debris and high resolution number distributions can be used to calculate the number of viruses vs the number of virus aggregates. Estimating the concentration of viruses present is essential in understanding the loss of product at each step of the purification process (and hence can be used to optimise the process in terms of product yield) and also virus concentration is essential when trying to understand dosage in the final product.

Live vs. total viral titre and comparison with infectivity assays
The ability of the NanoSight technique to size and count a virus whether it is live or inactive allows the users to obtain an idea of the relative concentrations of infective particles vs. total particles when used in conjunction with infectivity assays. Infectivity assays such as plaque assays are the most widely used technique to estimate live viral titres in vaccine manufacture. To accurately quantify infectious viral titres, these techniques rely on a single virion infecting a single cell in a culture, subsequent replication and infection of surrounding cells causes a plaque to form which can then be quantified.

Clearly these assays require the virus to be infectious and as such are not applicable for inactivated vaccines. These assays do not account for viruses which have lost infectivity during the purification process in a live attenuated vaccine. As such, for live attenuated vaccines, a viral titre as calculated by an infectivity assay will vary considerably from the total viral titre as counted by the NanoSight technique. It is frequently found that the ratio of infective to non-infective particles can vary be 2 or even 3 orders of magnitude such that ratio of infective viruses is 1/1000 of the total particle content. This has clear implications, when understanding the efficacy of the manufacturing process and steps can be made to improve and optimise the product yield based on NanoSight data. Similarly upon administration of the final product, the presence of non-infectious viruses will also induce an immune response (as per an inactivated vaccine), a factor that needs to be considered when understanding dosage in the final product.

Understanding aggregation of viruses
The NanoSight technique measures particle size on a particle-by-particle basis and as such can generate high resolution particle size distributions. The size distribution can be used to estimate relative concentrations of monomeric vs. aggregated material due to the fact that the technique not only measures particle size but counts the number of particles of a specific size. The qualitative aspect of the technique also provides a quick insight into the state of aggregation, as can be seen in the images below. The first image shows a highly purified preparation of influenza virus whilst the second image shows a sample with a high degree of aggregation. Independent of a number distribution, the user can quickly and reliably understand the state of aggregation within a preparation.

Infectivity assays are unable to distinguish between aggregated and non-aggregated material in a viral preparation. A plaque forming unit may be a single virion or a single aggregate containing many potentially infective viruses. If administered aggregated viral material may de-aggregate in vivo and as such, an infectivity assay may grossly underestimate the infectious viral content within a preparation.

Summary

  • The NanoSight technique can be used as tool to quantify the total viral content and can be used alongside infectivity assays to calculate the relative concentrations of infectious to non-infectious viruses in a live attenuated vaccine
  • The NanoSight technique is ideally suited to calculate viral titres in inactivated vaccines - infectivity assays cannot be used for these vaccines
  • Viral concentrations are important when trying to understand product yield, immune response and potential allergenic reaction to the vaccine
  • The state of aggregation at each step of the manufacturing process and in the final product and be monitored and measured using the NanoSight technique
  • Infectivity assays have no ability to discriminate single virions from aggregated viral material
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