Immune Potentiation of PLGA Controlled-Release Vaccines for Improved Immunological Outcomes

Abstract

With the emergence of SARS-CoV-2 and the continued emergence of new infectious diseases, there is a need to improve and expand current vaccine technology. Controlled-release subunit vaccines provide several benefits over current vaccines on the market, including the use of less antigen and fewer boost doses. Previously, our group reported molecules that alter NF-κB signaling improved the vaccine's performance and improved adjuvant-related tolerability. In this report, we test how these immune potentiators will influence responses when included as part of a controlled-release poly(lactic-co-glycolic) vaccine formulation. Murine in vivo studies revealed that SN50 and honokiol improved antibody levels at early vaccine time points. Microparticles with SN50 produced strong antibody levels over a longer period compared to microparticles without SN50. The same particles also increased T-cell activity. All of the immune potentiators tested further promoted Th2 humoral responses already exhibited by the control CpG OVA microparticle formulation. Overall, under controlled-release conditions, immune potentiators enhance the existing effects of controlled-release formulations, making it a potentially beneficial additive for controlled-release vaccine formulations.

Figure 1

Analysis of controlled potentiator microparticles created by the double emulsion technique via SEM. (A) CpG OVA microparticles, (B) CpG OVA SN50 microparticles, (C) CpG OVA Honokiol microparticles, (D) CpG OVA Capsaicin microparticles (scale bar in all images is 10 μm). (E) In vitro release profile of microparticles containing CpG OVA Microparticles (black), CpG OVA SN50 (orange), CpG OVA Capsaicin (blue), or CpG OVA Honokiol (green) stirred in PBS at 37 °C over a 4-week period.

Figure 2

In vivo T-cell and antibody responses of controlled-release subunit vaccine containing SN50. (A) OVA-specific CD8 T-Cell responses measured by OVA^257–264MHC I from PBS (black bars), CpG OVA microparticles (green bars), CpG OVA unencapsulated (yellow bars), CpG OVA SN50 microparticles (blue bars), CpG OVA SN50 unencapsulated (purple bars). (B) OVA-specific CD4 T-Cell responses measured by OVA^323–339MHC II tetramers staining performed on lymphocytes harvested on day 21 (n = 5). Some samples were not present due to not harvesting enough lymphocytes for staining. (C) Serum day 28 anti-ovalbumin Ig’s (IgG + IgM + IgA) antibody levels measured from day 28 (n = 5). (D) Anti-ovalbumin Ig’s (IgG + IgM + IgA) antibody levels PBS (black), CpG OVA microparticles (green), CpG OVA Unencapsulated (yellow), CpG OVA SN50 Microparticles (blue), CpG OVA SN50 unencapsulated (purple) were measured over time from serum collected every 2 weeks after day 28 (n = 5), mean reported with standard deviation as the error. Statistics was performed with unpaired t test between CpG OVA Micro and CpG OVA SN50 Micro. One-way ANOVA was performed with Tukey’s test. *P < 0.05, **P < 0.01, and ***P < 0.001. Significance compared to CpG OVA microparticles. n.s., not significant.

Figure 3

In vivo performance of controlled-release subunit vaccine containing capsaicin and honokiol. (A) Percentage of OVA^257–264 MHC I positive tetramer staining from PBS (black bars), CpG OVA microparticles (green bars), CpG OVA Capsaicin microparticle (yellow bars), CpG OVA Capsaicin unencapsulated (blue bars), CpG OVA Honokiol microparticles (purple bars), CpG OVA Honokiol unencapsulated (red bars). (B) OVA^323–339MHC II positive tetramers staining on lymphocytes (n = 5). A few samples resulted in an insufficient number of lymphocytes for staining. (C) Day 28 anti-ovalbumin Ig’s (IgG + IgM + IgA) antibody levels measured from day 28 serum (n = 5). Mean reported with standard deviation as the error. One-way ANOVA was performed with Tukey’s test. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant.