Vaccine development is a long, intensive process that begins with the determination of public health needs and priorities. Several organizations have compiled priorities for research and development, including the World Health Organization/United Nations Development Program for Vaccine Development. There are several stages of vaccine development and testing, beginning with an exploratory stage to identify natural or synthetic antigens. Next, pre-clinical work begins and cell culture and animal models are used to determine the immunogenicity and safety of a vaccine candidate. Following the approval of an Investigational New Drug application to the US Food and Drug Administration, clinical trials may begin. Specialized assays are developed in the pre-clinical and early clinical trial phases, and are validated in later clinical trial phases. Prior to lot release, assays have well-characterized parameters and criteria, although the assay development process is not without challenges. The immune system is highly complex; however, new technologies and techniques offer solutions. Eventually, a successful vaccine candidate will progress through to Phase III trials and be licensed. The entire rigorous process can take upwards of 15 years to complete.
Immunogenicity, efficacy, exposure, and safety
First, it is important to understand the underlying mechanism of action. “Vaccines are generally aimed at modulating or priming the immune response to a pathogen so that the body can rapidly eliminate the pathogen and minimize disease,” explains Claire Richards, PhD, principal scientist, infectious disease, discovery services at Charles River Laboratories (Wilmington, MA). Pre-clinical vaccine research includes assay development, in part, for the quantification of the immune response. Rafiqul Islam, MS, executive director of bioanalytical services at Celerion Inc., echoes the importance of immunogenicity assays: “Multiple assays are required to measure immune responses to provide confidence that the candidate vaccine is at least capable of eliciting a robust immune response.”
Islam provides insight into the clinical development of vaccines, which “requires a specific set of specialized assays to demonstrate the immunogenicity, efficacy, exposure, and safety. The development and validation of these assays requires integration of therapeutic insights, state-of-the-art technologies, custom reagents, and robust quality management systems.”
Which assays, then, are used to assess the immune response? “A suitable assay to determine immune stimulation is usually required along with possible inhibition assays to show the immune response can reduce the growth of the pathogen; [for example] antigen-specific ELISAs, inhibition ELISA, neutralization assay, [and] effective concentration to give 50 percent survival (EC50),” says Dr. Richards. Table 1 outlines a summary of the assays commonly used to support vaccine development. The humoral, or antibody-mediated, immune response relies on B-cell recognition of antigens and subsequent antibody release. Ligand-binding techniques, such as ELISA (enzyme-linked immunosorbent assay), are often used to assess the humoral response. Conversely, the cell-mediated immune response relies primarily on T cells and is often assessed with ELISpot (enzyme-linked immunospot) or flow cytometry.
|Intended Purpose||Technology Platform||Assay Description|
|Immunogenicity Assessment—Humoral Antibody Response||Ligand-binding assay (e.g., ELISA, ECLIA)||Measurement of total antibody|
|Cell-based assay (e.g., viral-neutralization assay, bacterial-killing assay)||Functional antibody assay measurement|
|Immunogenicity Assessment—Cell-mediated Immunity||ELISpot, flow cytometry||Measurement of immune cells' response upon exposure to specific antigens|
|Efficacy||Ligand-binding assays and molecular assays (e.g., qPCR)||Measurement of antigen|
|Exposure and Safety||Cell-based assays and molecular assays||Measurement of infectivity and biodistribution|
The immune response assays may be used to determine next steps in a pre-clinical study. According to Dr. Richards, “The immune response prior to infection may be assessed, and if a suitable response has been induced, the models can be challenged or receive a further vaccine dose.”
Additionally, specific assays are required to assess vaccine efficacy, exposure, and safety (Table 1). Efficacy assays are designed to measure antigens, using “ligand-binding assays and molecular assays (e.g., qPCR),” says Islam. Both cellbased and molecular assays are used to assess infectivity, a measure of exposure and safety. The plaque-forming assay is a commonly used infectivity assay involving cell monolayers overlaid with agar to prevent the virus from spreading in the medium. As cells propagate infection, plaques begin to form. “At the end of a study, bacterial or viral load in organs may be assessed by determining the number of colony-forming units (CFUs) or plaque-forming units (PFUs),” says Dr. Richards.
It is essential to select the optimal assays for vaccine development, and there are several different technologies to compare. “Assays developed to support vaccine studies should be automation friendly, as vaccine trials require analysis of a large number of samples,” notes Islam. Numerous automated liquid handling and cell culture systems are available and dramatically reduce the amount of time spent performing tedious tasks as well as improving consistency.
“The most widely used technology [involves] ligandbinding assays and cell-based assays,” says Islam. Ligandbinding assays such as ELISA rely on the formation of an immune complex between the antibody and antigen. Cell-based neutralization assays offer a functional antibody measurement, as they are designed to detect antibodies capable of inhibiting viral replication.
While these assays are widely used, “the trend currently is to collect and evaluate more assay parameters using a divergence of technologies,” says Islam. “Technologies such as flow cytometry, ELISpot, qPCR, gene expression arrays, and multiplexing assays are gaining popularity, as each one of them provides unique insight into the safety, efficacy, immunogenicity, and biodistribution of vaccines.” In the clinical realm, a T-cell ELISpot, for example, is used to quantify the number of reactive antigen-specific T cells, indicative of a patient’s immune response.
Related Article: Adapting to the Needs of Cell-Based Assays
Selecting the appropriate assay for vaccine development is sometimes a difficult task. According to Islam, “The choice of the most relevant assay that correlates with protection against infection or disease is challenging due to the fact that immune responses are polyclonal and multidimensional by nature.” The human immune response is certainly complex. Polyclonal B-cell activation is characterized by production of numerous different antibodies against a single antigen, which may be both advantageous for the host and play a possible role in the development of autoimmune disease. Given this complexity, assay selection during vaccine development requires numerous considerations.
Scientists at Charles River outline several considerations to ensure a well-designed assay, ranging from availability of control compounds to robustness and scalability. Reagents, for example, are especially important when developing an assay for vaccine research, as they require significant time to produce. “In most cases, the generation of reagents requires immunization of animals or identification of large batches of reactive PBMCs [peripheral blood mononuclear cells], which are time-consuming,” says Islam. Given the complexity of assay design, it may be useful to consult an assay-development service for expertise and guidance.
Vaccine development is a complex process that presents several unique challenges. From the pre-clinical perspective, Dr. Richards notes that “inducing an effective response to the vaccine, optimizing the assays to ensure sensitivity, and ensuring the disease model is appropriate and reproducible” are all challenges associated with the process.
The intricacies of the immune response pose unique challenges for assay development, notes Islam, particularly for assessing parallelism in the context of a polyclonal response. “In order for immunological assays to generate reliable immune response data, it is important that the dose response curves of the samples be parallel to the reference curve. However, in most cases, due to the varying clonal compositions of the antibody response from different subjects, parallel dose response curves cannot be expected,” Islam explains. Further difficulty lies in the “demand for multi-parameter assays or multiple assays for the same analyte,” says Islam. He provides an example of measuring T-cell phenotypic markers such as cell surface markers, intra-cellular markers, etc., along with functional markers such as cytokine production.
Vaccines are designed to harness the power of the immune system to identify and eliminate disease-causing pathogens, and applications have expanded from infectious disease control to include cancer and autoimmune disease. Each vaccine is developed based on the immune response to a pathogen, which is a highly complex process. As such, there are numerous challenges in the assay development and screening process for vaccines. New techniques including flow cytometry, ELISpot, qPCR, gene expression arrays, and multiplexing assays are being implemented to overcome these challenges. Further, as cell culture and liquid handling automation technology continues to improve, so to does assay efficiency. Advancements in this field have enormous potential for improving human health, beyond what we once dreamt possible.