Fig 7. Immunization with a combination of Ad35.HSV.gD2 and Ad35.HSV.gB2 shows enhanced protection compared with individual components after HSV-2 intravaginal challenge.

Female BALB/c mice were immunized intramuscularly once at T = 0d with Ad35.HSV.gD2, Ad35.HSV.gB2 (10¹⁰ vp/mouse, N = 10/group), Ad35.HSV.gD2/ Ad35.HSV.gB2 (10¹⁰ vp/vector, N = 10/group) or mock immunized with formulation buffer (N = 10). Mice were intravaginally challenged 6 weeks after the first immunization (T = 42d) with 200LD50 (3.2x10⁴ pfu/mouse) WT HSV-2 G strain and monitored daily for (a) survival and (b) clinical scores over 21 days after infection. (c) Viral shedding was assessed by plaque assay on vaginal swabs collected on days 1, 2 and 4 after challenge (viral titers expressed as log₁₀ plaque forming units (pfu)/mL). HSV-2 VNT (titer expressed as log₂ IC₅₀), gD2 ELISA and gB2 ELISA (titer expressed as log₁₀ EU/mL) were performed on sera collected 1 day before challenge and correlation analyses were performed between (d) HSV-2 VNT vs viral shedding (Area Under the Curve, AUC) per individual animal on Day 1, 2, 4 (Day 1–4), (e) HSV-2 VNT vs gD2 ELISA titers and (f) HSV-2 VNT vs the sum of gD2 and gB2 ELISA titers. Dotted lines indicate the limit of detection (LOD) and horizontal lines represent mean value per group. Statistical comparisons of viral shedding between immunized groups (c) were performed by a pairwise Wilcoxon test with a 6-fold Bonferroni adjustment. Results of statistical analysis are depicted by asterisks: **p<0.01, *** p<0.001, ns: not significant. Correlation analyses were performed using the Spearman rank correlation method with the correlation coefficient and two-tailed p value for each analysis depicted in the figure.