Fig. 7: GpepIP primary immunization prior to trimer immunization elicits functional antibody production.

a Immunization scheme. BALB/c mice were primed three times (with a 3-week interval) by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Subsequently, all groups were immunized with the clade A BG505 gp140 NFL trimer adjuvanted with Alum for three times (with a 3-week interval). Sera were collected 7 days after each trimer immunization (post 1–3). b BG505-specific IgG production was examined in all three groups post each trimer immunization by ELISA using a serum dilution 1:400. c Antisera from all three groups after the third trimer immunizations were analyzed for IgG subclass switching by ELISA using a serum dilution 1:1600. d The neutralizing activity (neutralization 50% inhibitory dilution (ID₅₀)) of antisera from adjuvant, GpepIP, and pepIP primary followed by three BG505 booster immunizations were tested against tier 1 and tier 2 HIV-1 viruses via a TZM-bl cell-based neutralization assays. MLV-pseudotyped virus was used as negative control for non-HIV-1-specific inhibitory activity in the assay. Antibody CH01-31 was used as positive control (shown as antibody concentration). e BG505 was chemically labeled with fluorescein isothiocyanate (FITC) and incubated with BMDCs at 37 °C for 2 h. Cells were then collected and antigen uptake was measured by flow cytometry. To evaluate antisera for their function, fluorophore-labeled BG505 was pre-incubated with antisera used in e at different dilutions before adding into BMDCs. The uptake rate is defined as FITC-positive cells compared to no BG505 (medium) group. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b n = 6 for pre-bleed; n = 2 independent experiments for post 1–3. n = 4 and 3 independent experiments for c and e, respectively. P-values were determined using Student’s two-sided t-tests. Source data are provided as a Source Data file.