Phosphate-mediated coanchoring of RBD immunogens and molecular adjuvants to alum potentiates humoral immunity against SARS-CoV-2

Coanchoring a SARS-CoV-2 immunogen and molecular adjuvants to alum extends vaccine kinetics and amplifies humoral immunity.

: Antigen sequences. The amino acid sequences of RBD antigens used in these studies. (A) RBD antigens were expressed with terminal cysteines which can be coupled to short peptide linkers consisting of an N-terminal maleimide group and C-terminal pSer residues separated by a 6-unit poly(ethylene glycol) spacer. (B) pSer-modified RBD antigens are anchored to alum via ligand exchange between the phosphates in the pSer residues and hydroxyls on the surface of alum. (A) RBDJ antigens with pSer4 or pSer8 peptides conjugated at the N-terminus were assayed for phosphates per protein by a malachite green assay. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. (B) Unmodified, pSer4-, or pSer8conjugated RBDJ were mixed with alum, and the fraction of protein bound to alum was assessed before ("Loading") and after incubation for 24 hours in 10% mouse serum at 37°C. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. (C) Unmodified, pSer4-, or pSer8-conjugated RBDJ were mixed with alum and incubated in 10% mouse serum at 37°C. The fraction of protein bound to alum was assessed longitudinally. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test.
(D) A modified sandwich ELISA approach was used to analyze the antigenicity profile of pSermodified RBDJ. Shown are binding profiles of hACE2-Fc (top left), CR3022 (top right), H4 (bottom left), and B38 (bottom right) to RBDs captured on alum-coated plates (n=3 replicates), and the area under individual binding curves (E). Statistical significance was determined by twoway ANOVA followed by Sidak's multiple comparison test. Values plotted are means ± standard deviation. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. immunized with 10 µg unmodified, pSer4-, or pSer8-conjugated RBDJ and 100 µg alum, and germinal center (GC) responses in draining inguinal lymph nodes were analyzed by flow cytometry over time for total GC B cell counts (top), RBD-specific GC B cell counts (middle), and percent RBD-specific GC B cells (bottom) at 9, 14, 21, and 28 days post-immunization. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test.

Fig. S4. pSer-RBDJ drainage is a combination of antigen-alum complex trafficking and release of antigen from alum at the injection site.
(A) BALB/c mice (n=5 animals/group) were immunized with 10 µg unmodified, pSer4-, or pSer8-conjugated RBDJ and 100 µg alum and boosted at 6 weeks. Half-maximal inhibitory titers (ID50) values were assessed for hACE2-RBD interactions at day 35 and day 70. The dashed line indicates the limit of detection. Values plotted are geometric means ± geometric standard deviation. Statistical significance was determined by two-way ANOVA followed by Sidak's multiple comparisons test. (B) Mice were immunized with 10 µg fluorescently labeled RBDJ plus alum and the fluorescence at the injection site was quantified longitudinally (n=4 animals/group), as in Fig. 3A-B. The signal remaining at the injection site at day 49 for pSer8-RBDJ was subtracted from the longitudinal pSer8-RBDJ signal and plotted for comparison to pSer4-RBDJ. Values plotted are means ± standard deviation. Statistical significance between pSer4-RBDJ and pSer8-RBDJ was determined by two-way ANOVA followed by Tukey's posthoc test. (C) Mice were immunized with 10 µg RBDJ plus 100 µg of labeled alum and the fluorescence at the injection site was quantified longitudinally (n=3 animals/group). Values plotted are means ± standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. geometric means ± geometric standard deviation. Statistical significance was determined by twoway ANOVA followed by Tukey's post-hoc test. (C) Mice (n=5 animals/group) were immunized with 10 µg unmodified or pSer4-conjugated RBDJ and varying amounts of alum, and germinal center (GC) responses in draining inguinal lymph nodes were analyzed by flow cytometry at day 14 post-immunization. Values plotted are means ± standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test.
(D) Alum loaded with AlexaFluor647-labeled pSer4-RBDJ were mixed with alum labeled with pSer4-AlexaFluor488 at low density and incubated together for 2 days prior to imaging (left). Representative image shown. The fluorescence overlap was assessed (right) and (E) the fraction of alum particles with fluorescent signal colocalization was measured. Values plotted are means ± standard deviation. (F) Mice (n=5 animals/group) were immunized with 10 µg pSer4-RBDJ plus 200 µg alum at varying average antigen densities. Immunizations were prepped by first loading pSer4-RBDJ on alum at the indicated ratios and then supplementing alum just prior to immunization such that all groups received an equal alum dose. GC responses in draining inguinal lymph nodes were analyzed by flow cytometry at day 14 post-immunization. Values plotted are means ± standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (G) Mice (n=5 animals/group) were immunized with 10 µg pSer4-RBDJ plus 200 µg alum at varying average antigen densities. Serum IgG antibody responses were assessed longitudinally by ELISA. Arrows indicate immunization time points. Values plotted are geometric means ± geometric standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. Statistical comparisons between all pSer4-RBDJ groups are denoted above plot, while statistical comparison between each pSer4-RBDJ group against the RBDJ group is denoted between pSer4-RBDJ groups and RBDJ group. (H) Serum SARS-CoV-2 pseudovirus neutralizing titer ID80 (PSV NT80) were assessed for serum collected at day 28 and day 56. The dashed line indicates the limit of detection. Values plotted are means ± standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. S6. Co-adjuvants SMNP and CpG promote balanced antibody isotype responses and enhance humoral responses.
(A) BALB/c mice were immunized with 10 µg fluorescently labeled RBDJ with or without alum plus co-adjuvants CpG or SMNP, and the fluorescence at the injection site was quantified longitudinally (n=3 animals/group). Values plotted are means ± standard deviation. Statistical significance between pSer4-RBDJ groups was determined by one-way ANOVA followed by Tukey's post-hoc test. (B) Mice (n=5 animals/group) were immunized with 10 µg unmodified RBDJ or pSer4-RBDJ and 100 µg alum and/or 30 µg CpG or 5 µg SMNP and boosted at 6 weeks, as in Fig 4E-  Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test.

Fig. S7. Co-adjuvants enhance antigen uptake and germinal center responses
BALB/c mice (n=5 animals/group) were immunized with 10 µg AlexaFluor555 labeled antigen and 100 µg alum and 5 µg SMNP or 30 µg CpG, and the inguinal lymph nodes were collected 7 days post-immunization. (A) Representative flow cytometry gating plots. (B) The number of cells positive for AlexaFluor555 labeled antigen is plotted for B cells, monocytes, neutrophils, subcapsular sinus macrophages, medullary macrophages, and dendritic cells. Values plotted are means ± standard deviation. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. (C) Mice (n=5 animals/group) were immunized with 10 µg unmodified RBDJ or pSer4-RBDJ and 100 µg alum and/or 5 µg SMNP and the RBD-specific