(M1130-06-36) A Cell-To-Cell Contact Immune System Mimicking In Vitro Overlay Assay: Evaluating Nanoparticle Vaccine Formulations for Adaptive Immunity Responses

Purpose: T lymphocyte (T-cell)-mediated adaptive immune responses, in conjunction with innate immune responses, contribute to the intricate complexity of the immune system. In the process of initiating immune responses, antigen recognition, and internalization by antigen-presenting cells (APCs), and subsequent presentation of the processed antigen with conserved epitopes to the T cells via major histocompatibility complexes (MHC) I and II play a crucial part. Transiently stimulated T cells exhibit an atypically rapid succession of extensive cell divisions, differentiating into cytotoxic CD8+ T cells and long-lasting memory CD4+ T cells. Thus, established immunological memory persistently retains the capability to re-engage in proliferation upon encountering subsequent immunological stimuli. Ensuring the activation and proliferation of T lymphocytes specific to a pathogen is a critical aspect of vaccine development, particularly in the initial formulation phases. Evaluating vaccine candidates for the same at this stage is key to optimizing formulations, enhancing efficacy, and prolonged protection. Our in-house formulated vaccines employ microparticles (MPs) and nanoparticles (NPs) for efficient vaccine delivery. We recognized the need to establish a high throughput, inexpensive in vitro assay that mimics the in vivo immune environment to establish cell-to-cell contact between APCs and T cells. This assay aims to facilitate cell-to-cell contact between antigen-presenting cells (APCs) and T cells, enhancing antigen presentation and promoting T cell activation against the specific vaccine antigen. We hypothesize this cell-to-cell contact overlay setup will facilitate sufficient antigen presentation to the T cells, thus leading to subsequent clonal expansion of the T cells against the vaccine antigen. An in-vitro T cell proliferation assay using Carboxyfluorescein succinimidyl ester (CFSE) was optimized to study and measure the expansion of lymphocyte divisions in response to the nanoparticulated vaccine candidates. We aim to optimize the assay in the dendritic cells (DCs) – T cells overlay model, wherein after stimulation with the vaccine particles, successful antigen presentation by the DCs to the CFSE-stained T cells can occur. We also aim to study the extent of T-cell proliferation in response to various concentrations of both bacterial (different strains of N. gonorrhoeae) and viral (measles, zika, and H1N1) vaccine candidates. The proliferated T-cells are quantified using flow cytometry, thus evaluating the efficiency of our vaccine candidates and qualifying them for further pre-clinical studies.

Methods: Briefly, DCs were exposed to treatment groups of vaccine MPs, adjuvanted MPs & blank MPs. We plated 50,000 DCs per well and exposed them to vaccine MPs for 24 hours, after which, the particles were removed from the wells. 250,000 CFSE-stained T cells were overlaid on the stimulated DCs and were co-cultured for 6 days. These co-cultured plates were incubated at 37 °C, 5% CO2 during the experiment. T cell proliferation was monitored daily for 6 days using Flow cytometry analysis. Comparisons were made using T-test (non-parametric) analysis for statistical representation. We tested the following vaccine formulations: Two gonorrhoea prototype vaccines consisting of bovine serum albumin (BSA) microparticles (MPs) loaded with inactivated Neisseria gonorrhoeae, a flu prototype vaccine consisting of poly D, L-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with inactivated H1N1 virus, inactivated Zika virus and measles virus. We also included adjuvanted particles loaded with Addavax® and Alum.

Results: Our findings demonstrate that T cells exposed to N. gonorrhoea prototype vaccines BSA MPs + adjuvanted MPs proliferated, generating daughter cell populations with decreasing dye intensities over time. Initially, the parent population had significantly higher CFSE dye intensity than the daughter population across all groups. However, by day 6, the parent population decreased significantly while the daughter population increased. Notably, the N. gonorrhoea vaccine + adjuvanted MPs treated group exhibited significantly higher cell proliferation compared to blank control groups. Similarly, T cells treated with viral vaccine candidates against measles, H1N1 flu, and zika vaccine PLGA NPs + adjuvanted NPs also proliferated and generated daughter cell populations with decreasing dye intensities from day 0 to day 6. The parent population initially had significantly higher CFSE dye intensity than the daughter population across all groups, but by day 6, the parent population diminished significantly, and the daughter population increased. Furthermore, the H1N1 flu vaccine NPs and adjuvanted NPs treatment group showed significantly higher daughter cell proliferation compared to blank control groups. Additionally, concentration profiles were established by measuring T cell proliferation in response to various vaccine candidate concentrations.

Conclusion: We successfully tested the robustness and adaptability of the immune-mimicking overlay assay by testing it against various bacterial and viral vaccine candidates and establishing their concentration profiles. The studies demonstrate that when exposed to our nanoparticulate vaccine formulations, DCs induce T-cell clonal expansion against specific antigens. Our future studies aim to test the assay in assessing T cell clonal expansion upon antigen presentation in the presence of cytokines like IL-2 and to record the T cell repertoire upon subsequent challenge by the live antigen in vitro.

References: 1. Terrén I, Orrantia A, Vitallé J, Zenarruzabeitia O, Borrego F. CFSE dilution to study human T and NK cell proliferation in vitro. Methods in Enzymology, vol. 631, Elsevier; 2020, p. 239–55. https://doi.org/10.1016/bs.mie.2019.05.020.
2. Bagwe P, Bajaj L, Gala RP, D‘Souza MJ, Zughaier SM. Assessment of In Vitro Immunostimulatory Activity of an Adjuvanted Whole-Cell Inactivated Neisseria gonorrhoeae Microparticle Vaccine Formulation. Vaccines 2022;10:983. https://doi.org/10.3390/vaccines10070983.

Acknowledgements: The project is funded by an NIH grant 1R15AI133473-01A1
Graphics are created using Biorender.com


Dendritic cell (DC) uptake microparticles and T-cell interaction: Representative images of the uptake of microparticles by antigen-presenting dendritic cells and T cell interaction observed using the fluorescence microscope (A). The FITC-BSA microparticles were seen to be rapidly taken up by the dendritic cells and were accumulated in the cytosol and vacuolar compartments. The activated dendritic cells in the presence of DAPI-stained T cells (B&C) show T cell interaction with the dendritic cells(B) and cell division of the activated T cells (C).


Graphical representation of the cell-to-cell contact immune mimicking overlay assay in determining the T cell proliferation in response to vaccine candidates.


Representation of T cells proliferation across days 0-6 upon overlay with APCs that are pulsed with vaccine NPs.