*2.9. ELISA*

The HIV ELISA was performed using 1 μg/mL HIV consensus C gp120 (Immune Technology Corp., New York, NY, USA) in PBS with 0.5% Tween 20 (PBS-T). After a blocking step, serum was diluted to 1:50 and then 4-fold from there in 1% FBS in PBS-T. Each sample was run in duplicate. After a 1 h incubation, plates washed and incubated with goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA) at a 1:5000 dilution in 1% FBS in PBS-T. Plates were then developed as described above, and the OD450 values were obtained.

Avidity Index ELISA: The avidity of the humoral response was assessed against universal hemagglutinin at 10 days post final vaccination for influenza studies. Plates were coated with 1 μg/mL of hemagglutinin ((H1N1) (A/New Caledonia/20/99) Immune Technology Corp., New York, NY, USA) in PBS. After a blocking step, serum was diluted to 1:50 or, for the dilution curves, 1:50 and then 4-fold from there in 1% FBS in PBS-T. Each sample was run in duplicate with half of the wells treated and half left untreated. After a 1-h incubation, plates were washed five times with PBS-T. Half of the wells for each sample were incubated with denaturing reagent (8 M urea) for 5 min while the others were incubated with PBS. Plates were washed and incubated with goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) at a 1:5000 dilution in 1% FBS in PBS-T. Plates were then developed as described above, and the OD450 values were obtained. The avidity index was determined by dividing the OD450 values of the treated samples by those of the untreated samples and multiplying by 100.

## *2.10. Statistical Analysis*

Statistical analysis was performed using a one-way modified ANOVA with a Turkey post-hoc test for immunogenicity studies and Mantel–Cox test for challenge studies. All analysis was performed using GraphPad Prism Software (La Jolla, CA, USA). Horizontal bars represent mean with error bars expressing the standard error. *p*-Values < 0.05 were considered as statistically significant.
