2.12. Alhydrogel®‘85’, Aluminum Hydroxide Oxide Gel [AlO(OH)], Adjuvant Dose Ranging

Three groups of female BALB/c mice (10 mice per group) were immunized by subcutaneous (SC) needle injection with Alhydrogel^®‘85’ formulated RBD-C-tag antigen at day 0 (20 μg RBD-C-tag), day 20 (20 μg RBD-C-tag), and day 41 (20 μg RBD-C-tag) (Figure 2a). In group 1, the 0.2 mL volume of each inoculation formulation of Alhydrogel^®‘85’ [AlO(OH)] contained a total of 320 μg Alhydrogel^®‘85’ and 20 μg RBD-C-tag; in group 2, the formulation contained a total of 500 μg Alhydrogel^®‘85’ and 20 μg RBD-C-tag; and in group 3, the formulation contained a total of 640 μg Alhydrogel^®‘85’ and 20 μg RBD-C-tag was used. Alhydrogel^®‘85’ alone was used as a negative control. At days 19, 27, 40 and 51 blood was collected and sera were evaluated for anti-RBD-C-tag immunoglobulin and virus neutralizing antibody titers.

Vaccination of BALB/c (H-2^d) female and K18-hACE2, C57Bl/6J (H-2^b) female mice with affinity purified C1-RBD-C-tag adjuvated with Alhydrogel^®‘85’: (a) three groups of BALB/c mice (10 per group) were injected subcutaneously (SC), with 20 μg of C1-RBD-C-tag/mouse adjuvanted with 320 μg, 500 μg, or 640 μg Alhydrogel^®‘85’. As negative placebo controls, three groups of three mice were vaccinated with 320 μg, 500 μg, 640 μg Alhydrogel^®‘85’ alone. All groups were primed and double boosted at 20 and 41 days post-prime, respectively; and (b,c) two groups of eight individual female K18-hACE2 C57BL/6J mice were primed and boosted either once (b) or twice (c) with 20 μg RBD-C-tag/mouse with 640 μg Alhydrogel^®‘85’ at day 21 (b) and at days 21 and 42 (c). As negative controls, two groups of three individual K18-hACE2 C57BL/6J mice were each vaccinated with 640 μg Alhydrogel^®‘85’ alone in the same manner as in (b,c). At 20 days post-injection with the 1 boost (b) and 14 days post the 2 boost (c) all C1-RBD-C-tag and control vaccinated mice were anesthetized and challenged Intra-Nasally (IN) with 2000 PFU of live SARS-CoV-2/mouse. General observations for monitoring morbidity and weight loss of the live virus challenged mice were carried out for three weeks post-challenge, and the final blood collection of all surviving mice was performed.