Figure 5. BLyS treatment prior to immunization increases the serum neutralizing Ab responses.

(A) Sera from mice immunized twice (2x) with Env in adjuvant collected 21 days after the second immunization were analyzed for total Env-binding Abs (left panel) and for neutralizing activity against a panel of heterologous HIV-1 isolates (right panel). OD50 values are shown for total Env-specific Ab titers. Neutralization data are shown as the reciprocal dilution giving 50% neutralization (ID50 titer). (B) The same analysis as in (A) using sera from animals immunized three (3x) or four times (4x) with Env in adjuvant collected 4 days after the last immunization. Each point in the ELISA analysis represents data from one mouse and the bar shown in (B) indicates the mean of all values ±SD. (C) The frequency of PBS- and BLyS treated mice with neutralization activity against MN, HXBc2, SF162 or 6535, combining all experiments (2xEnv, 3xEnv and 4xEnv), were plotted and analyzed by a non-parametric Mann-Whitney test. To have an equal number of mice in each group one mouse was removed from the 4xEnv BLyS group. This mouse (number 46) displayed the highest ID50 neutralization titer against several of the viruses. Thus, by removing this mouse from the comparison we under-estimate rather than over-estimate the effect caused by the BLyS treatment. (D) The potency of neutralization was also analyzed by calculating the average ID50 neutralization titers against MN, HXBc2, SF162 and 6535 among PBS- and BLyS-treated mice, combining the three experiments (2xEnv, 3xEnv and 4xEnv). Statistical significance was analyzed using the non-parametric Mann-Whitney test. The ELISA data from 2x are from one experiment with 12 mice/group; the results from 3x are representative data from two independent experiments with 5–7 mice/group, and the data from 4x are from one experiment with 4–5 mice/group. The neutralization data are from one experiment each for 2x (12 mice/group), 3x (7 mice/group) and 4x (4–5 mice/group).