PEG modified liposomes containing CRX-601 adjuvant in combination with methylglycol chitosan enhance the murine sublingual immune response to influenza vaccination

Abstract

The mucosa is the primary point of entry for pathogens making it an important vaccination site to produce a protective mucosal immune response. While the sublingual (SL) mucosa presents several barriers to vaccine penetration, its unique anatomy and physiology makes it one of the best options for mucosal vaccination. Efficient and directed delivery of adjuvants and antigens to appropriate immune mediators in the SL tissue will aid in development of effective SL vaccines against infectious diseases. Herein we demonstrate a robust immune response against influenza antigens co-delivered sublingually with engineered liposomes carrying the synthetic Toll-like receptor-4 agonist, CRX-601. Liposome modification with PEG copolymers (Pluronics), phospholipid-PEG conjugates and chitosan were evaluated for their ability to generate an immune response in a SL murine influenza vaccine model. Phospholipid-PEG conjugates were more effective than Pluronic copolymers in generating stable, surface neutral liposomes. SL vaccination with surface modified liposomes carrying CRX-601 adjuvant generated significant improvements in flu-specific responses compared with unmodified liposomes. Furthermore, the coating of modified liposomes with methylglycol chitosan produced the most effective flu-specific immune response. These results demonstrate efficient SL vaccine delivery utilizing a combination of a muco-adhesive and surface neutral liposomes to achieve a robust mucosal and systemic immune response.

Keywords: Chitosan; Influenza; Liposomes; Mucosal; Sublingual; TLR4; Vaccine.

Figure 1. Stability of adjuvant-liposomes in presence methylglycol chitosan (MGC)

Size/PDI and ζ-potential values with increasing concentration of MGC for adjuvant-loaded unmodified, PE-PEG2K and PE-PEG5K modified liposomes (A) & (C); and Pluronic L64, F68 and F127 modified liposomes (B) & (D). For (A) & (B), sizes are plotted as bars on the left Y-axis and PDI values as dot plot on right Y-axis. Data are expressed as mean ± SD, (n = 3). Particles in the μm size range tended to precipitate over time.

Figure 2. Flu specific HI titers and mucosal IgA following SL vaccination with phospholipid-PEG modified liposomes

(A) Serum HI titers and (B) mucosal wash anti-flu IgA concentration from mice vaccinated with the indicated formulations. Serum, tracheal wash, and vaginal wash samples were collected from mice on day 56 following vaccination on days 0, 21 and 42. The horizontal dashed line represents the titer necessary for seroconversion. Anti-flu IgA concentration in vaginal wash (VW) and tracheal wash (TW) samples are indicated as black circles (●) and grey squares (■), respectively. Values less than the lower limit of quantitation (LLOQ) at 1:20 sample dilution for HI titers and 1:100 dilution for IgA are represented as a value of 1. Data expressed as mean ± standard error (n = 8). **, P < 0.005 compared to unmodified liposome group; n.s., not significant (P > 0.05) compared to IM group.

Figure 3. Flu specific HI titers and mucosal IgA responses following SL vaccination with Pluronic modified liposomes

(A) Serum HI titers and (B) mucosal wash anti-flu IgA concentration from mice vaccinated with the indicated formulations. Serum, tracheal wash, and vaginal wash samples were collected from mice on day 56 following vaccination on days 0, 21 and 42. The horizontal dashed line represents the titer necessary for seroconversion. Anti-flu IgA concentration in vaginal wash and tracheal wash samples are indicated as black circles (●) and grey squares (■), respectively. Values less than the LLOQ at 1:20 sample dilution for HI titers and 1:100 dilution for IgA are represented as a value of 1. Data expressed as mean ± standard error (n = 8). n.s., not significant (P > 0.05) compared to unmodified liposome treatment group.

Figure 4. Flu specific titers following SL vaccination with MGC or CO coated phospholipid-PEG modified liposomes

Serum HI titers from mice vaccinated with the indicated formulations. Serum samples were collected from mice on day 56 following vaccination on days 0, 21 and 42. The horizontal dashed line represents the titer necessary for seroconversion. Values less than the LLOQ at 1:20 sample dilution are represented as a value of 1. Data expressed as mean ± standard error (n = 8). **, P < 0.005, compared to unmodified liposome group; *, P <0.05, compared to 10 μg CO + PE-PEG2K liposome group.

Figure 5. Flu specific titers following SL vaccination with MGC coated Pluronic F127 liposomes

Serum functional antibody HI titers from mice vaccinated with the indicated formulations. Serum samples were collected from mice on day 56 following vaccination on days 0, 21 and 42. The horizontal dashed line represents the titer necessary for seroconversion. Values less than the LLOQ at 1:20 sample dilution are represented as a value of 1. Data expressed as mean ± standard error (n = 8). ***, P < 0.0005 compared to unmodified liposome group; n.s., not significant (P > 0.05) compared to unmodified liposome treatment group.