March 1, 2010 (Vol. 30, No. 5)

New Instrument Counts and Sizes Viruses and Their Aggregates in Liquid Suspension

There are two main categories of viral vaccines—live (attenuated) 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 an extended period of time. The majority of successful viral vaccines fall under this category including vaccines for measles, mumps, rubella, influenza, yellow fever, and polio (Figure 1).

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 also in assessing the concentration of virus material present. This is where the NanoSight instrument (Figure 2) adds real value. The particle-by-particle approach to sizing and counting viruses can 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. This information can be used to optimize the process in terms of product yield. Virus concentration is also essential when trying to understand dosage in the final product.

Figure 1. A model virus: Viruses typically range from 20 nm to 300 nm.

Live vs. Total Viral Titer

The ability of the NanoSight technique to size and count a virus whether it is live or inactive allows 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 titers in vaccine manufacture. To accurately quantify infectious viral titers, 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 measuring the final product for inactivated vaccines. These assays do not account for viruses that have lost infectivity during the purification process in a live attenuated vaccine. As such, for live attenuated vaccines, a viral titer as calculated by an infectivity assay will vary considerably from the total viral titer (i.e., infectious and noninfectious) as counted by the NanoSight technique.

The ratio of infective to noninfective particles can frequently vary by two or even three orders of magnitude such that the ratio of infective viruses is 1/1,000 of the total particle content. This has clear implications for understanding the efficacy of the manufacturing process. Steps can then be taken to improve and optimize the product yield based on NanoSight data. Similarly, upon administration of the final product, the presence of noninfectious 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.

Figure 2. The goal of the NS500 is to provide an easy-to-use, reproducible platform.

Understanding Virus Aggregation

The NanoSight technique measures particle size on a particle-by-particle basis and can generate high-resolution particle-size distributions. The size distribution can be used to estimate relative concentrations of monomeric vs. aggregated material because 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 quick insight into the state of aggregation, as can be seen in Figure 3. Figure 3A shows a highly purified preparation of influenza virus, while Figure 3B 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 nonaggregated material in viral preparations. A plaque-forming unit may be a single virion or a single aggregate containing many potentially infective viruses. Aggregated viral material may de-aggregate in vivo upon administration and, as such, an infectivity assay may grossly underestimate the infectious viral content within a preparation.

Figure 3: A highly purified preparation of influenza virus (A) and a sample with a high degree of aggregation following freeze-thawing (B).

Virus-Like Particles

Virus-like particles (VLPs) are a relatively new area within vaccine development. They have been designed to overcome the problems associated with certain recombinant protein vaccines that have poor immunogenicity resulting from poor presentation of the viral antigens to the immune system. This can be overcome to a certain 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. VLPs have been shown to have significant potential in eliciting a stronger and lengthier immune response than more traditional recombinant protein vaccines.

In terms of characterizing such structures, infectivity assays cannot be used as VLPs, are devoid of the RNA or DNA required for replication, and hence, they are noninfectious. In addition, they cannot be quantified using qPCR again due to the fact that they do not contain DNA or RNA. The NanoSight technique does not require the particle to be infectious nor contain DNA/RNA and hence represents an attractive method of characterizing these structures.

The NanoSight technique can be used as a tool to quantify the total viral content and can be used alongside infectivity assays to calculate the relative concentrations of infectious to noninfectious viruses in a live attenuated vaccine. This method is ideally suited to calculate viral titers in inactivated vaccines where infectivity assays cannot be used to characterize the final product.

Andrew Malloy ([email protected]) is head of application sciences at NanoSight. Web:

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