In this review we discuss recent advances in evaluation of vaccine quality control (QC) and safety utilizing gene expression assays.
A brief history of vaccine development
Since Edward Jenner’s experiment in the late 1790s utilizing cowpox to confer immunity against smallpox, scientific research has led to the development of numerous types of vaccines to safely and effectively elicit immune responses that protect against infection. The first vaccines consisted of entire pathogens that had been killed or inactivated, or weakened through a process with chemical, heat or radiation treatment (also called attenuation) so that the delivered pathogen would not cause disease but would still elicit an immune response that could target proteins of a live, infectious pathogen. While these inactivated or live-attenuated pathogen-based vaccines can elicit strong protective immune responses, not every pathogenic microbe can be effectively targeted with these approaches.
Deeper biological understanding of pathogenic root causes, along with technical advances in cell and tissue culture methods, allowed for the field of vaccine development to improve and expand the uses of live-attenuated vaccines, as demonstrated most successfully by the measles, mumps and rubella (MMR) vaccine. While live-attenuated vaccines have proven to be successful for some viruses, complex viral pathogen and host interactions make universal vaccine approaches challenging, not to mention other non-viral pathogens like bacteria or parasites. To address challenges posed by various types of pathogens, other types of vaccine were developed that would help to better stimulate the immune system better and more specifically, such as subunit vaccines. Subunit vaccines only include immune response stimulating protein components of the pathogen, or antigens, which not only make them safer but also make them easier to produce. These types of vaccines usually require incorporation of an adjuvant, which helps to elicit a stronger protective immune response, as some antigens alone are not sufficient to induce adequate long-term immunity. Recent years have also seen the development of vaccines using recombinant technology such as those that use virus-like particles (VLP) and nucleic acid vaccines. VLP vaccines, are non-infectious and non-replicative, composed of one or more viral structural proteins that have the ability to self-assemble, and may be used as vehicles for drug delivery, gene therapy, and cancer treatment. More recent and promising vaccine development programs have focused on nucleic acid vaccines, which delivers the genetic material that encodes the immune eliciting antigen(s), thereby allowing the body’s own machinery to produce the antigen. This has many potential advantages that could improve large-scale manufacturing, provide excellent stability, and elicit a long-term immune response.
Why evaluate gene expression for vaccine QC and safety?
Vaccine safety assessment is an early and essential step in the process of vaccine development. Typically, vaccine safety testing (e.g., QC tests and adjuvant development) has been conducted in animal models, which can require a large number of animals and is a lengthy process. Additionally, traditional animal toxicity tests have some limitations in that the results cannot be extrapolated for factors related to adverse events seen in humans. One relatively new approach in vaccine safety is to identify gene expression biomarkers that are known to be indicators that are effective in evaluating safety. Expression profiling of biomarker genes has already been adopted for safety evaluation of synthetic drug development and biologics. If the biomarker gene expression levels are correlated with a high degree with immunotoxicity and immunogenicity, their expression levels may be superior predictable factors for vaccine and adjuvant safety. In addition, benefits of using gene expression biomarkers can include the time to results, safety assessment of vaccination administration route that can affect the potential toxicity of a vaccine formulation, and a mechanistic understanding of intra-patient differences in disease trajectories and the differences in important clinical outcomes.
Why QuantiGene assays?
The QuantiGene Plex assay offers researchers the beneﬁt of being able to study all relevant biomarkers simultaneously. Unlike other methods for gene expression analysis, this assay provides the unique benefit of being able to detect RNA directly in lysates or tissue homogenates without the need for purification or any enzymatic manipulation steps. An elegant example of using this assay for gene expression profiling and biomarker testing in vaccine safety is demonstrated in a paper published by Sasaki et al . In this study, the authors developed an 18-gene/transcript QuantiGene Plex panel for potential influenza safety testing based on previous microarray studies. Using this 18-plex panel, they assessed the safety of multiple administration routes using both vaccines and adjuvant solutions and comparing the biomarker gene expression profiles to a reference sample.
mRNA biodistribution for Nucleic Acid–Based Vaccines
mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development, and potential for low-cost manufacturing and safe administration. However, their application has been restricted by the instability and inefficient in vivo delivery of mRNA. Recently however, technological advances have largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans, specifically regarding optimized immunogenicity, mRNA translation, stability as well as delivery system. Although the manufacturing process for mRNA vaccines avoids the common risks associated with other vaccine platforms (like virus or constituent components), safety concerns remain around local and systemic inflammation (e.g., type I interferon responses), persistence of expressed immunogen, toxicity associated with nonnative nucleotides and delivery system components and biodistribution of (mRNA) vaccine. In a study published in Molecular Therapy, the authors used the QuantiGene Singleplex assay to quantify the amount of mRNA in serum and several tissues between 2 and 264hrs post intradermal dosing with the mRNA vaccine . The result from this study showed that irrespective of the route of administration the biodistribution was similar to an influenza virus inoculation.
mRNA vaccines are ushering in a new era of vaccinology. The ability to quickly generate candidates for vaccines has shortened the timeline for development. This facet has enabled the rapid development of candidate vaccines for SARS-CoV-2, which have shown early signs of promise. The use of powerful tools such as the QuantiGene assay allows researchers to rapidly address safety and quality concerns when developing these newer types of vaccines.
 Sasaki E et al. Modeling for influenza vaccines and adjuvants profile for safety prediction system using gene expression profiling and statistical tools. PLoS One. 2018 Feb 6;13(2):e0191896
 Bahl K et al. Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses. Mol Ther. 2017 Jun 7;25(6):1316-1327
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