Figure 6. Co-delivery of TLR-7/8a and protein antigen on a self-assembling thermo-responsive vaccine particle.

(a) Cartoon schematic of a thermo-responsive Poly-7/8a (TRPP-7/8a) modified with a coil peptide that forms heterodimers with a recombinant HIV Gag-coil fusion protein to form TRPP-7/8a-(CC)-Gag. Heterodimerization occurs at room temperature and particle formation results at temperatures greater than 33°C. (b) Temperature-dependent particle formation illustrated by dynamic light scattering. (c) Aqueous solutions of TRPP-7/8a-(CC)-Gag at 25°C and 37°C. (d, e) Co-localization of HIV Gag (labeled with anti-Gag PE) with TRPP-7/8a (labeled with carboxyrhodamine 110) was confirmed by (d) flow cytometry and (e) confocal microscopy. (f–i) BALB/c mice received subcutaneous administration of 50 μg of HIV-Gag coil formulated with either a control or TRPP-7/8a normalized for TLR-7/8a dose (1x dose = 2.5 nmoles, or 3x dose = 7.5 nmoles) at days 0 and 14. At day 28, DLN, spleen and serum from vaccinated mice were collected for analysis. Splenocytes were stimulated in vitro with an HIV Gag peptide pool. Antigen-specific IFNγ-producing (f) CD4 T cells (n = 5) and (g) CD8 T cells (n = 5) in the mixed splenocyte cultures, as well as (h) Tfh cells (n = 5) in draining lymph nodes were quantified by flow cytometry. (i) Serum was evaluated for anti-HIV Gag total IgG antibody titers (n = 5). In vivo studies are representative of two independent experiments. Data on linear axes are reported as mean ± SEM. Data on log scale are reported as geometric mean with 95% CI. Comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. CC = coiled-coil interaction.