Potent response of QS-21 as a vaccine adjuvant in the skin when delivered with the Nanopatch, resulted in adjuvant dose sparing

Adjuvants play a key role in boosting immunogenicity of vaccines, particularly for subunit protein vaccines. In this study we investigated the induction of antibody response against trivalent influenza subunit protein antigen and a saponin adjuvant, QS-21. Clinical trials of QS-21 have demonstrated the safety but, also a need of high dose for optimal immunity, which could possibly reduce patient acceptability. Here, we proposed the use of a skin delivery technology – the Nanopatch – to reduce both adjuvant and antigen dose but also retain its immune stimulating effects when compared to the conventional needle and syringe intramuscular (IM) delivery. We have demonstrated that Nanopatch delivery to skin requires only 1/100th of the IM antigen dose to induce equivalent humoral response. QS-21 enhanced humoral response in both skin and muscle route. Additionally, Nanopatch has demonstrated 30-fold adjuvant QS-21 dose sparing while retaining immune stimulating effects compared to IM. QS-21 induced localised, controlled cell death in the skin, suggesting that the danger signals released from dead cells contributed to the enhanced immunogenicity. Taken together, these findings demonstrated the suitability of reduced dose of QS-21 and the antigen using the Nanopatch to enhance humoral responses, and the potential to increase patient acceptability of QS-21 adjuvant.

was determined for each individual post vaccination serum, absorbance from both treated and untreated groups with 6M urea was determined. Percentage of urea resistant antibodies was calculated by dividing the optical density (OD) of the urea-washed samples by the OD of the unwashed samples, and percentage below 50% was considered low avidity IgG 45 .

IL-4 & IFNγ Enzyme Linked Immunospot (ELISPOT)
Interleukin-4 and interferon-gamma enzyme-linked immunospot (IL-4 and IFNγ ELISPOT) assays were performed to quantify antigen-specific cells producing IL-4 or IFNγ. This assay is well established and is based on previous study with slight modification 7 . Briefly, 7 days after vaccination, mice were euthanised by cervical dislocation and splenocytes were prepared. ELISPOT plates (Millipore MultiScreen-HA Cat#MAHAS4510) were coated with IL-4 or IFNγ capture antibody (MabTech Cat# 3311-3-250 or 3321-3; final concentration, 8 μg/ml). Influenza antigen was treated with 2.5% trypsin at 37 °C for 3 hours to release peptides. Splenocytes and treated influenza antigen (2 μg/ml) were mixed to stimulate cells in vitro at 37 °C in 5% CO 2 for 40 to 44 hours. An anti-IL-4 or anti-IFNγ biotinylated detection antibody (MabTech Cat# 3311-3-250 or 3321-6-250; final concentration, 1 Page 2 of 7 μg/ml) was added and incubated for 2 hours at room temperature. Avidin horseradish peroxidase (HRP; Sigma Cat# A-3151) was then added, and the plates were incubated for 1 hour at room temperature. The substrate DAB (SigmaCat#D0426)was prepared according to the manufacturer's instructions and added to the wells until spots appeared (5 to 8 minutes).The results reported were averaged between 3 technical replicates and with the background (no peptide) counts subtracted.
Total serum antigen specific IgG titres from three different doses of influenza antigen represented by  (6 ng , 60 ng  and 120 ng ) and × unimmunised mice was used as control. ELISA antibody data represent the Mean ± SEM, statistical significance is when p<0.05, n=5 C57BL/6 mice per group.
Statistical for endpoint titres were performed with one-way ANOVA. ng or 120 ng of influenza vaccine with or without 1.5 µg of QS-21 by the Nanopatch, or coadministering various dose (1.5 µg, 10 µg or 50 µg) of QS-21 with 120 ng of influenza antigen by needle and syringe intramuscular (IM) route. Nanopatch groups were represented by  unadjuvanted  and QS-21) and IM groups were represented by ,, and  (unadjuvanted  and of QS-21 1.5 µg,  10 µg or  50 µg). The number of spots was counted, averaged by 3 technical replicates and subtracted by the background (no peptide) wells. ELISPOT data represent the Mean ± SEM per million spleen cells, statistical significance is when p<0.05, one experiment of n=3 C57BL/6 mice per group. Statistical for endpoint titres were performed with one-way ANOVA.
Equivalent of antigen specific IL-4 producing cells are induced by all the doses of NP and IM groups, which supported the earlier data. However, IFNγ ELISPOT were producing background related data (sub-zero points), which is highly possible to be due to the lack of specific peptide during restimulation. The reason for this limitation was identified by Zhong et al 47 , influenza virus (PR8) contained major CD8 + T cells epitopes were located in nucleoprotein and polymerase acidic protein, which is not in the antigen (purified HA protein from A/California, A/Victoria and B/Wisconsin) used in this study. The presence of CD8 + T cells epitopes in HA protein were found to respond at a very low rate 47 and that different influenza strains may have different antigen presentation kinetics.
Alternative ways developed by Ingulli et al 48 , used ovalbumin-expressing influenza virus to infect their mice models and CD8 + T cells epitope from ovalbumin (SIINFEKL) for re-stimulation to detect influenza specific CD8 + T cell responses, which is a technique not available to our group.