The COVID-19 pandemic renewed interest in vaccinations, and scientific advancements abound. Developers are continually working to create vaccines that elicit targeted, strong, and long-lasting immune responses, protecting patients against viral and bacterial infections.
Clinical trials, which sometimes include hundreds or even thousands of volunteers, establish that a vaccination is both safe and effective.
The capacity to accurately evaluate the strength and persistence of an immune response to a pathogen is important to the success of any vaccination. Synexa presents a general overview of bioanalytical methods to vaccine studies, including the essential assays required and the challenges surrounding their development and practical implementation.
The goal of a vaccine
Vaccine candidates may include attenuated live viruses, inactivated viruses, toxoids, viral vectors, and mRNA vaccines. Vaccine technology is continually evolving, with new improvements in delivery techniques, adaptability, and immunogenicity.
Despite the wide range of techniques, all vaccines have the same goal: to stimulate the host immune system to produce a particular and long-lasting immunological response. This entails first activating indigenous T and B cells to create a population of responsive effectors capable of directly targeting foreign pathogens or releasing suppressive antibodies.
Key readouts
Therefore, the main bioanalytical readouts in a vaccine clinical study are as follows:
- The humoral response: Specific binding of pathogenic proteins or neutralizing antibodies
- Cellular response: Activation of pathogen-specific immune cells, primarily T-cells
Although each of these important readouts can be approached in a variety of ways, they are frequently intricate and customized, requiring that they be unique to the vaccine.
Humoral immune response assay considerations, build, and complexities
The identification of the humoral response generally depends on two main types of assays:
- Ligand binding assays: Identify and measure antibodies that bind specifically to the foreign antigen. These assays primarily consist of immobilizing an antigen on the surface of a well, followed by incubation with sera that contain binding antibodies, and concluding with a general detection step
- Neutralization assays: Identify antibodies that are functionally significant and inhibit the entry of a viral particle
While seemingly simple in theory, there are several difficulties that require an experienced approach to address:
Availability of positive control sera: Determining specificity or whether a response is “positive” necessitates a precisely calibrated cut point and positive control. Access to sera that have such antibodies is critical. Analytical laboratories can use a variety of methodologies, including international standards (where available), convalescent sera from exposed individuals, and trial participants who are already vaccinated.
In many circumstances, developing a successful test requires an experienced analytical lab and a collaborative approach with the vaccine developer.
The dynamic range of the assay: Several modern vaccinations contain many antigens (multivalent), allowing them to trigger a wide range of immune responses. As a result of this wide immunological response, measuring it might be difficult. Creating a suitable pool of positive sera that encompasses the spectrum of responses to various antigens is difficult and crucial.
The assay must identify both very high and low response rates. The capacity of an assay to detect these reactions is attributed to its dynamic range and linearity. Both are important criteria to consider when developing and validating an experiment. Furthermore, the assay design for a multivalent vaccination requires multiplexing of the immune response, which in turn requires the use of platforms such as MSD.
Containment of live viruses in neutralization assays: Neutralization assays identify functional antibodies that prevent viral entrance into a target cell. As predicted, this necessitates an in vitro test and a method of detecting viral entrance. Plaquing experiments have traditionally used live virus (typically labeled) and target cells, as well as a visual detection technique (commonly microscopy). These tactics are frequently utilized when the virus's entrance mechanism is not understood.
Unfortunately, these assays are less sensitive and often use a live virus, requiring varying levels of containment. When feasible, analytical laboratories use pseudovirus particles, fluorescently labeled virus-like particles that mimic the virus’s activity and entry. Bioanalytical laboratories can use the pseudovirus neutralization assay (PNA) to assess fluorescence in target cells and determine how neutralizing antibodies may limit viral entry.
Cellular immune responses and degrees of complexity
Cellular immune responses are objectively assessed using assays that measure cellular features, such as flow cytometry and ELISpot. The ELISpot test is one of the most frequent and technically less difficult ways for determining the frequency of cytokine-secreting cells. It works by trapping cytokines generated when immune cells (most often PBMCs) are stimulated with pathogenic antigens.
ELISpot assays are reliable and reproducible, and can detect low responders; however, their simple design limits what can be inferred from the results. ELISpot tests can detect up to two distinct cytokine responses and are unable to identify the contributing cell population. Despite this, ELISpot assays are commonly employed in vaccine studies, particularly when more complicated flow cytometry assays are not feasible.
In vaccine studies, flow cytometry can be used to detect activation-induced marker (AIM) expression, T-cell proliferation, peptide-MHC multimers, and intracellular cytokine staining (ICS). Synexa Life Sciences uses ICS as the primary example of a functioning cellular immune response assay.
ICS assays require stimulating a population of immune cells (often PBMCs) with a foreign pathogen, followed by multiparameter flow cytometry measurement of intracellular cytokine production as a sign of activation. The panels are often large and contain a variety of phenotypic, cytokine, and activation markers. These assays are very difficult to develop, verify, and generate massive data sets.
Concerns about their complexity have limited the use of ICS assays; nonetheless, skilled bioanalytical laboratories that understand the essential concerns in assay development can assist in providing rich and nuanced data sets. Few assays give more precise insights into the various immune responses to a foreign antigen, which are particularly valuable to developers. Some of the complications in ICS assay development include issues regarding:
- Representative samples: ICS assays, like humoral assays, rely on samples that closely resemble the cellular responses predicted in clinical samples. It can be difficult to get such samples, which need either prior exposure to the pathogen or prior vaccines. A tight collaboration between the lab and the developer is critical to guaranteeing the inclusion of relevant samples for validation
- Sample preparation: Proper sample preparation will be critical to an assay's performance. In most cases of ICS, PBMCs are the preferred sample matrix. It is critical that the collecting site can robustly isolate PBMCs with sufficient quantities and viability for downstream experiments
- Assay controls: Complex assays such as ICS require multiple controls to ensure cells behave consistently and appropriately. Assays may also comprise many duplicates, depending on the number of conditions and sample availability (PBMC counts), as well as internal quality controls, which are frequently made up of healthy PBMC samples and used to assure assay consistency throughout the period of clinical study
- Instrumentation and panel design: Harmonizing the expectations of data outputs with an appropriate instrument and panel is critical for validating a robust assay. It is critical for laboratories to be aware of the constraints of certain apparatus while also collaborating with developers to create panels employing fluorophores with minimal spectral spillover
- Sample batching efficiency and addressing analysis bottlenecks: Understanding throughput and sample batching is critical for large studies to ensure optimal turnaround time and resource use. Mitigations for PBMC thawing (which might be a key bottleneck for analysis) and plate-based analysis to boost throughput are two examples
The examples provided here are not exhaustive and do not apply to every scenario. Each trial and vaccine is unique, and collaborating with an experienced bioanalytical lab that understands what needs to be addressed when developing complex yet data-rich assays will go a long way toward ensuring a successful experiment.
Conclusions
Creating a clear and well-defined bioanalytical methodology to quantify humoral and cellular immune responses is critical for a successful vaccination study. There are several techniques, each with its own difficulties and implications. Navigating how the assays are administered and verified demands an experienced and adaptable provider.
At Synexa, vaccine research is in their DNA, and they are particularly proud of the complex, specialized assays they have validated for customers. This comprises two multiparameter ICS assays and a multiplexed humoral assay used in two large clinical studies of infectious diseases during the last two years.
About Synexa Life Sciences
Synexa Life Sciences is a biomarker and bioanalytical lab CRO, specializing in the development, validation and delivery of a wide range of complex and custom-designed assays.
With a team of over 200 staff across three global laboratory locations; Manchester, Turku (Finland) and Cape Town, we provide innovative solutions to support our customers to achieve their clinical milestones.
Our main areas of expertise include biomarker identification and development, large and small molecule clinical bioanalysis, (soluble) biomarker analysis (utilizing MSD, LC-MS/MS, ELISA, RIA, fluorescence and luminescence-based technologies), cell biology (including flow cytometry, ELISpot and Fluorospot) and genomic services to support clinical trials and translational studies.
We pride ourselves on our deep scientific expertise and ability to tackle complex problems, translating them into robust and reliable assays to support clinical trial sample analysis.
Since 2019, Synexa has been backed by Gilde Healthcare, a specialized healthcare investor. Synexa, improving the quality of human health through innovative biomarker and bioanalytical solutions.
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