vaccines). These tests are generally based on the binding of key protective antigens

in the vaccine to specific antibodies.

The progress in biosensor technology (e.g., surface plasmon resonance and

biolayer interferometry) has now enabled the evaluation of the quantity and quality

of vaccine antigen binding to antibody.

4.4.2.1.3

Live-Attenuated Vaccines

For live-attenuated vaccines (measles, mumps, rubella, varicella, oral polio vac-

cines), the efficacy of each vaccine lot is linked to the number of live viral particles

that can be detected by cell-based assays.

Techniques directly quantifying the number of viral particles (e.g., transmission

electron microscopy, ViroCyt, and Nanosight) have also been developed, but are

still limited to specific applications and are only utilized in the release testing of a

few vaccines. Moreover, these methods make no assessment of the viability of the

virus particles.

Thus, the most common methods used to quantify viruses are based on cell

infectivity and detection of cytopathic effects (e.g., for measles, mumps, and var-

icella vaccines). Serial-dilution methods in cell cultures (generally in microplates)

may involve identifying the end-point dilution––the minimum titer necessary to

cause infection––and the calculation of the dilution, leading to 50% cell infection

(TCID50) [31]–[33]. However, these methods may result in a significant variability.

Plaque assays are based on the assumption that a single infectious virus particle

infects one cell and its propagation leads to the surrounding cells also being infected,

producing a delimited zone of cytopathology (plaque). The number of plaques is hence

considered to represent the number of viable virus particles in the sample dilution.

The appropriate design of the plaque assay [34] reduces the assay variability [35]

but the assay can be compromised if the virus is poorly lytic or the culture con-

ditions are not correctly optimized (as with a semi-solid culture layer), potentially

resulting in limited virus propagation and plaque identification. Alternative readouts

based on the detection of single infected cells [36], [37] or the detection of nucleic

acid resulting from virus replication [38] are increasingly being used to improve

precision and increase throughput.

Finally, more refined methods [39], [40] combining the quantification and pre-

cise identification of the vaccine components are being developed, and may have

the potential to identify the potency of the viral particles.

4.4.2.1.4

Live-Vectored Recombinant Virus

General methods described previously can also be applied for the detection of

vaccines based on live recombinant virus platforms (e.g., lentivirus and adenovirus

platforms). However, a combination of a potency test with an evaluation of the

expression of the inserted sequence coding for the antigen is preferred.

4.4.2.1.5

Validation

Although during assay development, an evaluation of the assay performance and

suitability is sufficient, the potency test has to be well defined and qualified for

Phase III clinical studies and fully validated for consistency and release lots.

Cell lines for vaccine production

73