(M0930-02-14) T Lymphocyte Proliferation in Response to Microparticulate Vaccine Candidates against H1N1 Flu and Neisseria gonorrhea Pathogens

Purpose: Evaluating the capability of T lymphocyte (T-cells) proliferation by vaccine candidates as indicative of the development of adaptive immunity against the specific antigen, is important during the formulation stages. An in-vitro T cell proliferation assay, Carboxyfluorescein in succinimidyl ester (CFSE) assay, was established to study and measure the expansion of lymphocyte divisions. CFSE, a (5,6)-Carboxyfluorescein diacetate succinimide ester is a cell-permeant, non-fluorescent pro-dye that contains acetate groups. Upon removal of the acetate groups by the intracellular esterase, a highly fluorescent Carboxyfluorescein succinimidyl ester will be yielded and can be analyzed using Flow cytometry. As the cells divide and proliferate, the dye taken up by the cells is evenly distributed among the daughter cells during cell division. Each daughter T cell will have approximately half the level of CFSE as the parent T cell, leading to a progressive dilution of the fluorescent signal. Our vaccine formulations utilize microparticles (MPs) and nanoparticles (NPs) to deliver the vaccine candidates efficiently. By performing CFSE assay and establishing T cell proliferation in response to the vaccine candidates, we can screen and select the best vaccine formulation, further allowing them to progress to in-vivo pre-clinical testing. 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 hypothesize that the CFSE-stained T cells proliferate due to the antigen presentation by the DCs in this in-vitro setup. We also aim to measure and quantify the T cell proliferation in response to the vaccine candidates using Flow cytometry.

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 7 days. These co-cultured plates were incubated at 37 °C, 5% CO2 during the experiment. T cell proliferation was monitored daily for 7 days using Flow cytometry analysis. Comparisons were made using T-test (non-parametric) analysis for statistical representation. We tested the following vaccine formulations: A gonorrhea prototype vaccine consisting of bovine serum albumin (BSA) microparticles (MPs) loaded with inactivated Neisseria gonorrhea and secondly, a flu prototype vaccine consisting of poly D, L-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with inactivated H1N1 virus. We also utilized adjuvanted particles loaded with Cytosine phosphoguanine (CpG 7909), Addavax® and Alum adjuvants along with the vaccine MPs/ NPs treatment.

Results: Our results indicated that the T cells treated with Neisseria gonorrhea prototype vaccine BSA MPs + adjuvanted MPs proliferated to generate a series of daughter cell populations with lower dye intensities as days passed. On day 1, the parent population (high intensity of CFSE dye) was significantly higher than the daughter population (low-intensity CFSE dye) across all the treatment groups. However, as days passed, the parent population diminished significantly, and the daughter population increased significantly by day 7. Also, the N.gonorrhea vaccine + adjuvanted MPs treated group showed a significantly higher number of proliferated cells than the blank control groups, which are the cells only (DCs and CFSE-stained T cells) group and the blank BSA MPs treatment group. Similarly, T cells treated with H1N1 flu vaccine PLGA NPs + adjuvanted NPs, proliferated and generated a series of daughter cell populations with lower dye intensities from day 1 through day 7. On day 1, the parent population (high intensity of CFSE dye) was significantly higher than the daughter population (low-intensity CFSE dye) across all the treatment groups. Furthermore, a significant diminishing of the parent population and an increase in the daughter population was observed by day 7. Additionally, the treatment group with the H1N1 flu vaccine NPs and the adjuvanted NPs showed a significantly higher number of proliferated daughter cells than the blank control groups, which are the blank PLGA NPs treatment group and cells-only group (DCs and CFSE-stained T cells).

Conclusion: These studies with the two vaccine prototypes demonstrated that the DCs when exposed to nanoparticulate vaccine formulations, could induce T-cell proliferation against specific antigens. Our future studies aim to further advance the vaccine formulations that generate the best T cell proliferation, into in-vivo pre-clinical testing.

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.

Figure 1: Illustration of DCs- T cells CFSE overlay assay to estimate T cell proliferation in response to nanoparticulate vaccine formulations.

Figure 2: Flow analysis data illustrating proliferation of daughter T cells from parent T cells compared between Day 1 (black) and Day 7 (green) in gonorrhea vaccine BSA MPs + Adjuvanted MPs treatment group (left) and H1N1 flu vaccine PLGA NPs + Adjuvanted NPs treatment group (right).