Immune responses to HIV Env protein in sera from guinea pigs immunized with the rNDVs expressing gp160 (rNDV-gp160) by the i.n. route on days 0 and 21 in all the groups and boosted on days 49 and 79 with either rNDV-gp160 by the i.n. route in rNDV-gp160 group or purified gp120 and SOSIP gp140 proteins by the intramuscular (i.m.) route in the rNDV-gp160+gp120 and the rNDV-gp160+gp140 groups, respectively. HIV-1 gp120-specific total IgG (A), IgG1 (B), and IgG2a (C) serum antibody responses on days 0, 21, 28, 35, 42, 49, 56, 70, 90, 120, and 160 were analyzed by ELISA using purified gp120. ELISA titers were endpoint values and are defined as the last reciprocal serum dilutions at which the optical density was greater than 2-fold over the signal detected with the parental rNDV (control). Values plotted are the geometric mean titers ± standard errors of the means (SEM) for the 3 animals in each group. Antibodies specific to gp120 were not detected in any animal on any day in the parental rNDV control group. Arrows indicate times of rNDV and purified protein immunizations on days 0, 21, 49, and 79. Statistical differences between the results from all the groups were calculated by one-way analysis of variance (ANOVA). **, P value < 0.005; ***, P value < 0.0005.