(567a) Synthesis and Immunostimulatory Potential Evaluations of the Aluminum Oxyhydroxide-Poly(I:C) Combination Adjuvant

Polyriboinosinic acid-polyribocytidylic acid, Poly(I:C), is an artificial double-stranded RNA. After binding with TLR3, Poly(I:C) could induce the immunostimulatory effects. However, Poly(I:C) could be easily degraded both in vitro and in vivo, resulting inefficient targeting to specific cell populations or tissues, preventing the clinical transformation of Poly(I:C). In this study, we established a delivery system, i.e, a combination adjuvant. It is composed of Poly(I:C) and the rod-like aluminum oxyhydroxide (AlOOH), which is a type of aluminum salts that has been formulated in licensed human vaccines.

The combination adjuvant was prepared by mixing aluminum oxyhydroxide (AlOOH) nanoparticles and Poly(I:C) in saline. The concentration of Poly(I:C) in the formulation was varied from 0.2 to 2 mg/mL, and they were noted as Al-Poly(I:C)-L and Al-Poly(I:C)-H, respectively. The Poly(I:C) conjugation efficiency was 97.81±0.37% and 99.38±0.02% for Al-Poly(I:C)-L and Al-Poly(I:C)-H, respectively, and they exhibited good stability over a period of 40 days. TEM analysis showed that the pristine AlOOH nanoparticles exhibited classical rod-like shapes. With the inclusion of Poly(I:C), it did not change the rod-like shape of the pristine particles. As the concentration of Poly(I:C) was increased, aggregations appeared in the Al-Poly(I:C)-H formulation (Figure 1A). DLS measurement showed that the hydrodynamic size of pristine AlOOH nanorods in water was around 264±9 nm. For Al-Poly(I:C)-L and Al-Poly(I:C)-H combination adjuvants, their hydrodynamic sizes were increased, and they were 453 ± 19 and 804 ± 10 nm, respectively. The zeta potential of pristine AlOOH nanorods was 26±5 mV in water. The Al-Poly(I:C)-L posed positive charges, while the Al-Poly(I:C)-H carried negative charges in water, suggesting more negatively-charged Poly(I:C) were grafted on the surfaces of AlOOH nanorods. Fourier transform infrared spectroscopy (FTIR) also confirmed the inclusion of Poly(I:C) with AlOOH nanorods (Figure 1B).

In order to evaluate the enhancement of adaptive immune responses induced by combination adjuvants, female C57BL/6 mice (6-8 week) were administrated with Al-Poly(I:C) adjuvanted HBsAg VLPs via intramuscular (i.m.) injection on day 0 and 21 (Figure 1C). On day 42, serum was collected to determine the HBsAg-specific antibody titers. Combination adjuvants significantly promoted HBsAg-specific IgG, IgG₁ and IgG_2c titer compared with HBsAg VLPs (Figure 1D-F). The ratio of IgG_2c/IgG₁ for AlOOH-Poly(I:C)-H was around 1.9 (Figure 1G), suggesting a Th1-biased cellular immune response. To analysis the the cellular immunity induced by the combination adjuvants, spleen was isolated from immunized mice and further stimulated with HBsAg VLPs ex vivo. The combination adjuvants induced the activation of CD4⁺ T cells (CD4⁺CD69⁺) and CD8⁺ T cells (CD8⁺CD69⁺) (Figure 1H-I), as well as CD8⁺CD107α⁺ and CD8⁺FasL⁺, which are the indicators of cytotoxic T lymphocytes (CTLs) activation (Figure 1J-K).

In summary, this study has developed a combination adjuvant platform using the composed of Poly(I:C) and the aluminum oxyhydroxide. The combination strategy enables the development of adjuvants for both prophylactic and therapeutic vaccines.

Figure 1. Evaluation of the aluminum oxyhydroxide-Poly(I:C) combination adjuvant. (A) TEM images and (B) FTIR spectra of pristine AlOOH nanorods, Al-Poly(I:C)-L and Al-Poly(I:C)-H, respectively. (C) The immunization procedure of the HBsAg vaccination model. 8-week-old female C57BL/6 mice (n=6-7) were immunized via i.m. on day 0 and 21. On day 42, the blood serum was collected to determine the HBsAg-specific D) IgG, E) IgG₁ and F) IgG_2c titers. G) The ratio of HBsAg-specific IgG_2c to IgG₁. On day 28 and day 42, the activation of (H) CD4⁺ and (I) CD8⁺ T cells as well as the expression of the (J) CD107α and (K) Fasl on CD8⁺ T cells after ex vivo re-stimulation of cells with HBsAg VLPs (2 μg/mL) for 60 h. ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001 compared to saline buffer-treated mice; ^#p<0.05, ^###p<0.001 and ^####p<0.001 compared to HBsAg-treated mice; ^@p<0.05, ^@@p<0.05, ^@@@p<0.001 and ^@@@@p<0.001.