Because need for vaccines against some of the deadliest pathogens has never been greater, next-generation, cutting-edge efforts aimed at defining correlates of protection are eminently needed. While measurement of antibody titers provides a cursory impression of the overall magnitude of the humoral response, titers alone provide little insights into the quality of the antibody response, which may be key to uncovering a correlate of protection. To overcome this limitation, we have developed a number of high-throughput functional assays and high-throughput biophysical profiling of antibody isotype/subclass and glycosylation linked system biology/machine learning algorithms that collectively aim to define the specific characteristics of the most protective humoral immune responses in polyclonal pools of antibodies. Together, the functional and biophysical profiles provide an unprecedented depth of resolution of differences in pathogen-specific immune responses among different patient/vaccine groups, beyond simple changes in antibody titer. Moreover, due to the flexibility of these bead- and plate-based assays, a number of antigens can be simultaneously examined.
1. Fc receptor luminex array uses fluorescently coded microspheres to capture up to 500 antigen specificities simultaneously and profile the effector capacity of each antigen specificity by determining the ability of these antigen-specific antibodies to interact with Fc receptors, lectin-like molecules, innate immune receptors, and lectins, generating a high-dimensional biophysical data set. Antigens of interest (e.g., recombinant protein, whole virus, whole vaccine, or synthetic peptides) are covalently coupled to beads via primary amine conjugation or via streptavidin:biotin interactions. The beads are incubated with an antibody sample, allowing “on-bead” affinity purification of antigen-specific antibodies. The bound antibody is subsequently probed with tetramerized Fc receptors or lectins and assessed via Luminex. The data is reported as the median fluorescence intensity of a particular detector reagents for a specific bead channel. This assay has been GCLP qualified.
2. Antibody glycosylation is assessed on antigen-specific antibodies using antigen-coupled magnetic beads as customized affinity resins. Following antigen capture on these beads, the antibody Fc is enzymatically cleaved, and the glycans are enzymatically released, fluorescently labeled, and quantified by capillary electrophoresis. The data is reported as the percent of total glycans species. This assay has been GCLP qualified.
3. Antibody-dependent cellular phagocytosis (ADCP) assesses the ability of subjects’ antibodies which are presents in the serum/plasma to induce phagocytosis of antigen-coated fluorescent beads via Fc receptors. Streptavidin-conjugated beads are coated with biotinylated recombinant proteins of interest and subsequently incubated with subjects’ clinical material such as serum or plasma, which may contain antigen specific antibodies. A monocytic cell line (THP1) is mixed with beads and phagocytosis is allowed to proceed overnight. The extent of phagocytosis is measured by flow cytometry. The data is reported as a phagocytic score, which considers the proportion of effector cells that are phagocytosed and the degree of phagocytosis. This assay has been GCLP qualified.
4. Antibody-dependent complement deposition (ADCD) assesses the recruitment of complement component C3b on the surface of antigen-coupled beads. Beads are pulsed with antigens of interest and then incubated with subjects’ clinical material such as serum or plasma. Commercially available guinea pig complement is then added as a source of complement, and the level of C3b deposition is measured by flow cytometry. The data is reported as the complement deposition score, which considers the proportion of complement-positive beads, the degree of deposition, and the background (i.e., no antibody) values. This assay has been GCLP qualified.
5. Antibody-dependent neutrophil (ADNP) assesses the ability of subject antibodies to induce the phagocytosis of antigen-coated targets by primary neutrophils using primary neutrophils isolated from seronegative donors as effector cells. Fluorescent, streptavidin-conjugated beads are coupled to biotinylated antigens of interest, incubated with subjects’ clinical material such as serum or plasma, and overlaid on polymorphonuclear leukocytes (neutrophils) that are purified from fresh human blood. After a 1-hour incubation, the cells are fixed, and the amount of phagocytosis is assessed by flow cytometry. The data is reported as a phagocytic score, which considers the proportion of effector cells that are phagocytosed and the degree of phagocytosis. This assay has been GCLP qualified.
6. Antibody dependent NK cell activation (ADNKA) assesses antigen-specific antibody-mediated NK cell activation against protein-coated plates. Antibodies are added to antigen-coated ELISA plates. NK cells from seronegative donors are applied, and levels of activation markers and intracellular cytokines (MIP1b, IFNg, CD107a) are measured after 5 hours of incubation by flow cytometry. Additional subsets of effector cells, including unfractionated PBMCs, monocytes, and neutrophils can be tested. The data is reported as the percent of cells that express each of the markers alone and in combination. While not formally established as a GCLP-grade assay, a GCLP-like training program utilizing a standardized training set of samples and document-controlled SOPs have been implemented to reduce operator-to-operator and assay variability.
7. Antibody-dependent cellular cytotoxicity (ADCC) tests the ability of monoclonal antibodies to recruit NK cell lytic activity in a CEM based lysis assay. Target cells are pulsed with protein of interest and then pulsed and unpulsed are stained with a membrane and cytoplasmic dye. NK cells from healthy donors are added and incubated for 4 hours. Lysis is measured by flow cytometry and lysis is reported as the percent of live cells that have lost cytoplasmic dye. While this assay has been developed and standardized, it is not yet GCLP-qualified.
8. Antibody-dependent dendritic cell phagocytosis (ADDCP) assesses the ability of antibodies to induce phagocytosis of antigen-coated targets by DCs as well as DC activation/maturation and cytokine release. Antigen-coated beads are incubated with increasing concentrations of subjects’ clinical material such as serum or plasma and added to monocyte-derived DCs generated from seronegative donors. The extent of phagocytosis is measured via flow cytometry in conjunction with markers of cellular maturation (e.g., HLA-DR, CD83, and CD86). Additionally, supernatants are collected for cytokine profiling by Luminex (e.g., IL-10, IL-12p40, IFNa, IL-6, IL-8, etc). While this assay has been developed and standardized, it is not yet GCLP-qualified.
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