The Institutional Animal Care and Use Committee (IACUC) guidelines were followed with animal subjects tested in the immunization study. Seven-week-old BALB/c mice were purchased from the Jackson Laboratory. The mice were housed in ventilated cages in environmentally controlled rooms at TSRI, in compliance with an approved IACUC protocol and AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International guidelines. At week 0, each mouse was immunized with 200 μl of antigen/adjuvant mix containing 50 μg of antigen and 100 μl of the AddaVax adjuvant (InvivoGen) or 50 μl of the PIKA adjuvant (Yisheng Biopharma) as per the manufacturer’s instruction via the intraperitoneal route. At weeks 3 and 6, the animals were boosted with 50 μg of antigen formulated in the AddaVax or PIKA adjuvant. At week 8, the animals were terminally bled through the retro-orbital membrane using heparinized capillary tubes. Samples were diluted with an equal volume of PBS and then overlaid on 4.5 ml of Ficoll/Histopaque in a 15-ml SepMate tube (STEMCELL Technologies) and spun at 1200 rpm for 10 min at 20°C to separate plasma and cells. The plasma was heat inactivated at 56°C for 1 hour, spun at 1200 rpm for 10 min, and sterile filtered. The cells were washed once in PBS and then resuspended in 1 ml of ACK red blood cell (RBC) lysis buffer (Lonza). After two rounds of washing with PBS, peripheral blood mononuclear cells (PBMCs) were resuspended in 2 ml of BAMBANKER Freezing Media (Lymphotec Inc.). Spleens were also harvested and grounded against a 40-μm cell strainer (BD Falcon) to release splenocytes into a cell suspension. The cells were centrifuged, washed in PBS, treated with 10 ml of RBC lysis buffer as per the manufacturer’s specifications, and resuspended in BAMBANKER Freezing Media for cell freezing. One-third of the total serum per mouse, or 600 μl of serum, was purified using a 0.2-ml protein G spin kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Purified IgGs from four mice in each group were combined for characterization by ELISA binding and initial neutralization assays, while purified IgGs from individual mice in two nanoparticle groups, 6 and 10, were used for further analysis of HIV-1 neutralization in TZM-bl assays. Rabbit immunization and blood sampling were carried out under a subcontract at Covance (Denver, PA). Two groups of female New Zealand White rabbits, four rabbits per group, were immunized intramuscularly with 30 μg of the trimer or nanoparticle formulated in 250 μl of adjuvant AddaVax (InvivoGen) with a total volume of 500 μl, at weeks 0, 4, 12, 20, and 28. Blood samples, 15 ml each time, were collected at day −10 and weeks 1, 6, 14, 22, and 28, as shown in Fig. 6C. Plasma was separated from blood and heat inactivated for ELISA binding and neutralization assays.

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