Fig 1.
Neutralizing antibody responses correlate with protection against HSV-2 genital lesions.
(A) Serum neutralizing antibody titers are plotted from guinea pigs immunized with gD2. Serum was obtained after the third immunization. Animals are ranked from lowest to highest neutralizing antibody titer. (B) Cumulative lesion days per animal were monitored for 60 days post infection. Animals are ordered as in A. (C) The correlation between cumulative lesion days and serum neutralizing titers. P and rho (r) values were calculated by Spearman's correlation.
Fig 2.
ELISA antibody responses correlate with protection from HSV-2 genital lesions.
(A) ELISA endpoint titers are plotted from serum obtained after the third immunization. Animals are ranked from lowest to highest ELISA titer. Animal numbers are the same as assigned in Fig 1. The hatched bars represent animals with neutralizing titers ≥1:1280. (B) Cumulative lesion days per animal monitored for 60 days post infection. Animals are ordered as in A. (C) The correlation between cumulative lesion days and serum ELISA titers. (D) The correlation between neutralizing antibody titers and gD2 ELISA endpoint titers. P and rho (r) values in C and D were calculated by Spearman's correlation.
Fig 3.
Biosensor-based epitope binning.
(A) Properties of gD MAbs. MAbs that compete for binding are grouped into communities. Each community is sorted by color. The communities shown in A are reproduced with modifications from our prior publication by Cairns et al [30]. (B) Schematic of the biosensor based MAb competition assay. IgG from guinea pig #1 is incubated with gD2(285t) and flowed over the panel of MAbs printed on the biosensor chip. The gD2 fails to bind to one or more of the printed MAbs if IgG in the guinea pig serum competes with the printed MAb. The gD2 is stripped off the printed MAbs and the process is repeated with IgG from each of the other guinea pigs until all 25 have been evaluated. (C-I) Percent blocking of gD2(285t) binding to printed MAbs that recognize epitopes involved in receptor binding, activation of gH/gL and cell-to-cell spread. Each animal is assigned the same number as in Figs 1 and 2. Each guinea pig IgG was tested twice, except animal #11 was evaluated only once. Each prototype MAb was evaluated in both runs, except MAbs 1D3 and MC14 were tested once. Thus, 25 animals are in each group except that 24 animals are in the 1D3 and MC14 groups. Five MAbs were evaluated from the pink community, nine from the brown, including DL6 that is shown separately from the other brown community MAbs, three from blue, 2 from yellow, 1 from green and 2 from red. The IgG was considered positive for blocking if any blocking (≥1%) was detected. * indicates prototype MAbs within each community. Error bars represent SEM.
Fig 4.
Blocking cell-to-cell spread and binding to murine FcγRIII and FcγRIV receptors by prototype MAbs.
(A) MAbs MC16, DL6 and MC14 were added to HSV-2-infected HaCaT cells and plaque size was measured after 48 hours. R265 represents anti-gE2 polyclonal rabbit IgG used as a positive control. The dotted line represents plaque size in wells that contained media with no added antibodies. P values were determined by comparing plaque size in wells with added antibodies with the no-added antibody control wells. Statistics were calculated by Mann-Whitney; * p<0.05; **** p<0.0001; ns, not significant. (B-C) Binding of MAbs to murine IgG FcγRIV or FcγRIII that mediate ADCC. Endpoint titers were calculated based on the lowest MAb dilution that produced luminescence readings 2-fold higher than the no antibody control. Note that the Y-axis scales are different for B and C.
Table 1.
Properties of MAbs used for passive transfer in mice.
Fig 5.
Antibody passive transfer of prototype MAbs in mice.
BALB/c mice were passively immunized IP and infected the next day intravaginally with 5x103 PFU HSV-2 strain MS. Animals were observed for 10 days for survival (A), weight loss (B) and genital disease (C). Weights recorded are for surviving animals, which explains the increase in average weights observed in many groups towards the end of the experiment as euthanized animals drop out. Sample size: n = 15 for non-immune IgG; n = 10 for each prototype MAb; and n = 10 for uninfected animals. The Mantel-Cox method was used to calculate P values for survival curves. Each prototype MAb provided significantly better protection than non-immune IgG (P values noted on Fig 5A), and each prototype MAb provided significantly better protection than MC16 (p<0.001). Protection provided by DL11 or MC2 (both protected 80% of mice) was significantly better than MC23 (protected 30% of mice) (p = 0.036). No significant differences were detected for weight loss in any group. Significant differences between prototype MAbs and non-immune IgG for genital disease scores are noted in Fig 6C. Student’s t-tests were used to compare MAbs with nonimmune IgG for weight loss and disease scores. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.
Fig 6.
Correlation between the number of epitopes blocked and genital lesions.
(A) Number of epitopes blocked by IgG from each guinea. Animals are ranked from lowest to highest number of epitopes blocked. (B) Cumulative lesion days per animal monitored for 60 days post infection. Animals are ordered as in A. (C) Cumulative percent epitope blocking by IgG from each guinea pig. (D) Correlation between the cumulative lesion days and number of epitopes blocked. The P value was determined by Spearman's correlation. (E) Comparing animals with no lesion days with animals that had ≥1 lesion day. The P value was determined by the Students t-test. Horizontal bars in D and E represent means and SEM. (F) Correlation between the cumulative lesion days and the cumulative percent epitope blocking. The P value was determined by Spearman's correlation.
Table 2.
Lesion days based on antibodies produced to individual epitopes*.
Table 3.
Epitope-specific antibody responses in gD2 immunized guinea pigs and humans.