Demystifying the Immune Response3月 28th, 2017 / Hilary Graham
Developing tools to aid in the development of vaccines and therapeutic antibodies
Our understanding of the immune system has significantly progressed since Edward Jenner published his work in 1798 describing the development of the first vaccine. Jenner recognized that dairymaids previously infected with cowpox were immune to smallpox. To test his hypothesis, Jenner infected a young boy with a cowpox pustule from the hand of a milkmaid. Jenner then exposed the boy to smallpox, which he failed to contract. Jenner repeated the experiment on several children, including his son, finally concluding that the vaccination provided immunity.
Today’s vaccine developers are equipped with knowledge of the immune system workings as well as high throughput tools and computational technologies.
Tools of the Trade
Since it was invented in 1971 to understand immune cell interactions, the ELISA has been the gold standard for protein detection. Today’s complex research questions require more sophisticated tools that can simultaneously detect multiple analytes while conserving sample.
Margaret Ackerman, Assistant Professor in Thayer School of Engineering at Dartmouth College
Instead of focusing on research questions that surround a single disease state, Margaret Ackerman, Assistant Professor in Thayer School of Engineering at Dartmouth College, has dedicated a portion of her efforts to building novel, high throughput tools to evaluate the antibody response for vaccine development. Her group is specifically interested in improving methods of profiling the Fc region of antibodies, because that region serves as the link between the adaptive and innate immune systems.
Ackerman’s foundational Fc array platform assesses the ability of antibodies to bind to a suite of antigenic targets (e.g., peptides, proteins, and whole virus) and simultaneously defines Fc characteristics using a custom suite of antibody receptors and reagents. In characterizing the antigen specificities of clinical antibody samples (including collections of mucosal, blood, and stool products) and, in parallel, revealing the binding preferences of candidate Fc receptors, her Fc array provides a multi-dimensional profile of the humoral immune response. As these interactions are associated with known effector functions, these measurements can serve as a proxy for the functional capacity of an antibody response and, in turn, elucidate mechanisms of protection in vaccinated subjects. Ackerman’s group continues to expand the applications of the Fc array platform, including:
- Identifying antibody specificities for many different variants of a single viral protein with panels containing dozens of variants from different clades/classes (e.g., HIV gp120, Influenza HA), or antibody specificities for different viral serotypes by coupling whole virus to beads (e.g., Poliovirus serotypes PV1-3)
- Probing Fc interactions by determining antibody isotype, as well asinteractions with receptors and complement proteins
- Revealing receptor interactions that mediate immune system responses, such as ADCC, phagocytosis, complement-mediated destruction, neutrophil activation, dendritic cell uptake, and NK cell activation
From Discovery to Clinical Application
While many academic discoveries don’t make it out of the lab – Ackerman’s Fc array is now used by numerous academic and corporate collaborators to support vaccine trials.
In less than 2 years, Ackerman and Joshua Weiner established a GCLP (Good Clinical Laboratory Practices) facility and developed a qualified version of the Fc array to process clinical samples. The Dartmouth GCLP Antibody Laboratory (dAbL) is a true, high throughput facility equipped with two FLEXMAP 3D® instruments, an automated liquid handling workstation to facilitate work with 384-well plates, and three dedicated staff members – often running hundreds of samples and generating up to 100,000 antibody assessments each day.
To date, the dAbL has performed analysis of more than 30 primate and human studies in collaboration with private and public sectors and used this platform to characterize antibody responses to HIV, influenza, polio, rotavirus, and emerging pathogens in response to infection or vaccination. dAbL is just getting started. They plan to leverage their expertise for evaluating Fc binding interactions and the capabilities of the dAbL to develop additional assays to determine safety and efficacy of biologic therapeutics.
All photographs in this article are used with permission from Margaret Ackerman.
Multiplex Assay Development Tips from the Dartmouth GCLP Antibody Laboratory (dAbL)
Automation of the steps that lead up to analysis greatly increases consistency and reduces technician hands-on time. dAbL selected the Eppendorf epMotion® for their liquid handling and they also use liquid plate washers from BioTek.
High throughput techniques generate large data sets that can be challenging to manage. When designing your high throughput assay, be sure to plan for data analysis and storage. dAbL collaborated with scientists from the Computer Science Department to build a database complete with a web interface.
Good reagents are the foundation of a great assay. Whether you develop your reagents yourself or buy them from a trusted vendor, don’t assume they will work well in your assay format. Be sure to test each reagent when developing your assay to ensure high quality data and reproducibility.
Cultivate relationships. Collaborators can often provide access to high quality reagents not available for purchase.
Consider investing in an instrument service contract. A devoted service technician will understand you and your assay best, ensuring minimal downtime.
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