Fig. 2

Induction of increased antibody responses by BG505 SOSIP.664-ferritin in mice and rabbits. a Eight BALB/C mice were immunized three times (at weeks 0, 4 and 12) with either 2.8 μg of BG505 SOSIP.664 trimer or BG505 SOSIP.664-ferritin protein formulated with 25 μg MPLA adjuvant. The midpoint binding (EC₅₀) titers to BG505 SOSIP.664 trimer were determined at week 14 by Ni-NTA ELISA [2]; the median titers are denoted by horizontal lines. Statistical analysis was performed using a two-tailed Mann–Whitney U test. b Two groups of five New Zealand White rabbits received intramuscular immunizations at weeks 0, 4 and 12 with 200 μg of a non-adjuvanted DNA plasmid via electroporation of the quadriceps, followed by a protein boost at week 24 with 17 µg of protein in ISCOMATRIX™ adjuvant (75 units per rabbit) [24]. The DNA plasmids encoded either the soluble BG505 SOSIP.664 gp140 or the BG505 SOSIP.664 gp140-ferritin nanoparticles; none of the plasmids encoded furin. The protein boost was, correspondingly, either soluble SOSIP.664 trimers or SOSIP.664-ferritin particles, in both cases purified by a PGT145 bNAb column. The four historic control rabbits (indicated by circles in panel b) received identical DNA priming, but were then boosted with ISCOMATRIX™ adjuvanted (75 units per rabbit) soluble BG505 SOSIP.664 trimers (40 μg) that had been purified using 2G12-affinity chromatography followed by size exclusion chromatography (SEC) [2], which are antigenically identical to PGT145-purified BG505 SOSIP.664 trimers [25]. Anti-trimer serum binding titers over the course of the experiment were tested in D7324-capture ELISA using 2G12/SEC purified D7324-tagged BG505 SOSIP.664 trimers (0.5 μg/ml), essentially as described before [2, 9]. The medians of the midpoint binding titers (±error) are plotted. Asterisks indicate significant differences at specific time points (two-tailed Mann–Whitney U test; *P < 0.05). c Midpoint neutralization (IC₅₀) titers against the autologous neutralization-resistant (tier 2) virus, BG505, and against the negative control, MLV, at week 26. d IC₅₀ titers against a panel of heterologous neutralization-sensitive (tier 1A and tier 1B) viruses at week 26. The IC₅₀ titers in c and d were determined using the TZM-bl neutralization assay. The pre-bleed samples lacked neutralization activity (not shown). Neutralization assays were performed either at the Academic Medical Center (SF162, 6535.3, ZM197M, HXB2, DJ268.3, BaL, ZM109F, 94UG103, 92RW020, Q23env17 and MLV) or the Duke University Medical Center (DUMC) (BG505.T332 N, MN.3, MW965.26, Q259.d2.17, Ce1176_A3, Q769.d22, Q842.d12, YU2, Q23env17 and MLV). The fold difference in median IC₅₀ titer (horizontal lines) is depicted below the graphs. The dotted horizontal lines in the BG505 SOSIP.664 group represent the median titers for the five animals from the current experiment, i.e. excluding the four control sera. The titers were very similar when the four control sera were included or excluded. Statistical differences between the nine trimer-immunized rabbits and the five nanoparticle-immunized rabbits were determined using a two-tailed Mann–Whitney U test