Extended Data Fig. 9: pSer-MD39 binds to alum, enhancing humoral and neutralization responses.

(a) After incubation of MD39 trimer with alum for 30 minutes, 10% mouse serum was added, and solution was incubated for 24 hours. Binding of MD39 to alum was determined by the presence of MD39 in the supernatant after pelleting alum by centrifugation. MD39 concentration was measured by ELISA relative to a standard curve. (n=3 samples/group). Center lines and error bars represent mean and SD, respectively. (b) Representative images of fluorescently labeled MD39 or pSer₄-MD39 retention at the injection site after immunization with alum in BALB/c mice (n = 4 animals/group). (c) Quantification of IVIS images from (b). Data represents mean ± SD. (d) ISCOM-like saponin adjuvant does not inhibit pSer-immunogen binding to alum. Fluorescently labeled pSer₄-eOD (10 ug/mL) was combined with either alum or alum and saponin nanoparticles (labeled isco). After incubation for 24 hours in the presence of 10% mouse serum, the percentage of unbound eOD was measured by fluorescence. The fluorescence signal of pSer₄-eOD in the absence of alum was normalized to 100% (n=3 samples/group). Center lines and error bars refer to mean and SD, respectively. (e) BALB/c mice (n=10/group, pooled from two immunizations) were immunized with 5 µg MD39 or pSer₄-MD39 mixed with 50 µg alum and 5 µg saponin adjuvant. IgG titers from individual mice at week 6. . Statistical analysis by two-tailed Student’s t-test of the log-transformed data. Data are represented as mean ± SD of the log-transformed data. (f) Representative images of bone marrow ELISPOT plates for antibody-secreting cells at 3 months post immunization using the same conditions describe in (e). Experiments were performed twice, after two separate immunizations (n=5 mice/group for each experiment). (g) Quantification of mean bone marrow MD39 trimer-specific ELISPOT responses described in (f). Statistical analysis by two-tailed Student’s t-test (n=5 animals/group). Center lines and error bars refer to mean and SD, respectively. (h) Flow cytometry gating for identification of Env trimer-specific memory B cells. (i) Groups of BALB/c mice (n=5 mice/group for MD39 and pSer-MD39, n=2 mice/group for naive) were immunized were immunized with 5 µg MD39 or pSer₈-MD39 mixed with 50 µg alum and 5 µg saponin adjuvant on day 0. Shown are frequencies of MD39⁺ memory B cells from spleens at week 25. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. Center lines represent mean. (j, k) BALB/c mice (n=5 mice/group) were immunized with 5 ug antigen, and titers were measured four weeks after s.c. immunization. (j) Ser₄-eOD, alum-binding pSer₈-eOD, and eOD 60mer were used as antigens in combination with alum. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test (n=5 mice/group) of the log-transformed data. Center lines and error bars refer to mean and SD, respectively, of the log-transformed data. (k) MD39 control, pSer₄-MD39, and MD39-ferritin were used as antigens in combination with alum. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test (n=5 mice/group) of the log-transformed data. Center lines and error bars refer to mean and SD, respectively, of the log-transformed data. (l) BALB/c mice (n=10 mice/group) were immunized with 5 µg MD39 or pSer₄-MD39 mixed with 50 µg alum on day 0 with ISCOM-like adjuvant. His tag-specific IgG titers at day 63. Statistical analysis by two-tailed Student’s t-test of the log-transformed data. Center lines and error bars refer to mean and SD, respectively, of the log-transformed data. (m) Rabbits were immunized with MD39:alum or pSer₈-MD39:alum on weeks 0 and 8 (n=6 rabbits/group). Purified IgG antibodies from rabbit sera collected on week 10 was measured for neutralization against autologous tier 2 virus. Statistical analysis by Mann-Whitney test. Center lines and error bars refer to mean and SD, respectively.