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