Knowing viral concentrations increases our understanding of the behaviour of some vaccines. Andrew Malloy, head of Applications Sciences, NanoSight, explains how nanoparticle tracking analysis can quantify viral content
Figure 1: The equipment used for nanoparticle tracking
Knowing viral concentrations increases our understanding of the behaviour of some vaccines. Andrew Malloy, head of Applications Sciences, NanoSight, explains how nanoparticle tracking analysis can quantify viral content.
The ability 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 into two main categories: live (attenuated) vaccines and 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 a period of time.
The majority of successful viral vaccines fall into this 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.
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 understanding the concentration of virus material present.
This is where new technology that adds real value is needed. A 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 versus the number of virus aggregates. This new approach, called Nanoparticle Tracking Analysis, is provided in instruments from UK company, NanoSight (Fig. 1).
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). Virus concentration is also essential when trying to understand dosage in the final product.
The ability of the NanoSight technique to size and count a virus, whether it is live or inactive, allows the user to obtain an idea of the relative concentrations of infective particles versus 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 titers in vaccine manufacture. To quantify infectious viral titres accurately, 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 that can then be quantified.
Clearly these assays require the virus to be infectious and are not applicable for inactivated vaccines. These assays do not account for viruses that have lost infectivity during the purification process in a live attenuated vaccine. Virus binding affinity will also influence the infective viral titer as calculated by infectivity assays.
Changes in the experimental environment as well as lot-to-lot variability both in cell line and the specific virus tested can influence the viral binding affinity and resultant estimation of infective titer. In addition, infectivity assays do not account for the production of defective (i.e. inactive) viruses. These viruses may in fact elicit an immune response but will not be quantified by infectivity assays.
Taking all these factors into consideration, for live attenuated vaccines, a viral titre as calculated by an infectivity assay will vary considerably from the total viral titre as counted by NTA. It is frequently found that the ratio of infective to non-infective particles can vary by two or even three orders of magnitude, such that the ratio of infective viruses can be 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 NTA 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.
Virus-like particles (VLPs) overcome the problems associated with certain recombinant protein vaccines, in that they have a poor immunogenicity resulting from a poor presentation of the viral antigens to the immune system. This can be overcome to an extent through the addition of adjuvants, but perhaps another more attractive option is available through the creation of VLPs.
VLPs consist of an assembled structure of viral antigens creating a more authentic structure and conformation of the viral antigen. They have been shown to have significant potential in eliciting a stronger and lengthier immune response than more traditional recombinant protein vaccines.
In terms of characterising such structures, infectivity assays cannot be used as VLPs as they are devoid of the RNA required for replication and hence are non-infectious. Measurement of both particle size and state of aggregation as well as particle concentration is vital information in the characterisation of such products. The NanoSight technique represents a very attractive option for quantifying and sizing VLPs directly in liquid suspension as it does not require the particle to be infectious to measure it (Fig. 2).
Figure 2: Measurement of VLPs in solution
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 versus aggregated material due to the fact that the technique not only measures particle size but also counts the number of particles of a specific size.
Figure 3: (left) a purified preparation of influenza virus; (right) a freeze-thawed sample with a high degree of aggregation
The qualitative aspect of the technique also provides a quick insight into the state of aggregation (see Fig. 3). The left-hand image shows a highly purified preparation of influenza virus while the right-hand image shows a sample with a high degree of aggregation following freeze-thawing. Independent of a number distribution, the user can quickly and reliably understand the state of aggregation within a preparation through this qualitative assessment. (Fig. 4).
Figure 4: Qualitative assessment of the aggregated samples
Infectivity assays cannot 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 thus an infectivity assay may grossly underestimate the infectious viral content within a preparation.
NTA can be used to quantify the total viral content and used alongside infectivity assays to calculate the relative concentrations of infectious to non-infectious viruses in a live attenuated vaccine. It is also suited to calculating viral titers in inactivated vaccines for which infectivity assays cannot be used. 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 can be monitored and measured using NTA. Infectivity assays have no ability to discriminate single virions from aggregated viral material.
NTA is a light scattering method for nanoparticle analysis. It is being increasingly used for determining nanoparticle size through simultaneously tracking and analysing the trajectories described by a number of individual nanoparticles undergoing Brownian motion in a fluid.
The technique is centred on a sample analysis module which comprises a small metal housing containing a solid-state, single-mode laser diode (<30mW, 635nm) configured to launch a focused beam through the sample of liquid containing a dilute suspension of nanoparticles placed directly above a specially designed optical flat. The sample chamber has an approximate volume of 250µl and is 500µm deep into which the sample is introduced by syringe via a Luer port. The sample is left to equilibrate for 20sec prior to analysis.
Schematic of NTA light scattering method for nanoparticle analysis
The beam is caused to refract at the interface between the liquid sample and the optical element through which it is passed, such that it describes a path which is close to parallel to the glass-sample interface.
Particles resident in the beam are visualised by an optical microscope aligned normally to the beam axis and which collects light scattered from each and every particle in the field of view. A video of typically 20–60sec duration is taken of the moving particles.
The video is analysed by NanoSight’s proprietary analysis programme on a frame-by-frame basis, each particle being identified and located automatically and its movement tracked.
The thresholds for particle identification can be user adjusted, as can the gain and shutter speed settings of the camera, allowing the user to optimise the image for a particular sample type. The video sequence can be adjusted in terms of image smoothing, background subtraction, setting of thresholds, removal of blurring, etc, to allow particles of interest to be tracked without interference from stray flare or diffraction patterns, which can occasionally occur with non-optimum sample types.
Particles diffusing into the scattering volume are identified and followed for the duration of their presence in the beam or until they diffuse to within a certain distance of an adjacent particle at which point tracking is ceased, eliminating the possibility of analysing particle trajectories that cross behind each other. Movements of particles are followed through the video sequence and the mean squared displacement determined for each particle. From these values, the diffusion coefficient and hence sphere-equivalent hydrodynamic radius can be determined using Stokes-Einstein equation and with the results shown as a particle size distribution plot.