Figure 1.
Survival, following HSV-2 challenge, of mice immunized with codon-optimized polynucleotides encoding antigen and ubiquitinated antigen.
Mice sensitized with progesterone were challenged intra-vaginally with 50 (1.55×104 p.f.u.) or 500×LD50 of live HSV-2 strain 186, after three immunizations with 20 µg of plasmids expressing HSV-2 gD and/or ubiquitin-tagged truncated gD, and survival monitored. 10 mice/group/challenge dose were used (with the exception of the positive control which used 5 mice/challenge dose). Survival of mice immunized with a plasmid containing a codon-optimized insert encoding full length HSV-2 gD pcDNA3-O-gD2 [D], a plasmid encoding a ubiquitin-tagged truncated HSV-2 gD pcDNA3-O-Ubi-gD225–331 [UD], a mixture of D and UD [mix], a plasmid containing a codon de-optimized insert encoding full-length gD pcDNA3-W-gD2 [W], mouse thymidine kinase-deficient live HSV-2 strain 333 (5×105 p.f.u./mouse) [+ve], or empty pcDNA3 vector [-ve] is shown. For the 500× challenge, the survival differences between UD and D and between UD and mix were significant as determined by a log-rank (Mantel-Cox) test. The survival of W and -ve was significantly different from the vaccine and +ve control groups at both challenge levels. * P<0.05. This data has been published in patent US 2011/0287039 A1.
Figure 2.
HSV-2 DNA copy number in vaginal swabs, from immunized mice, taken after HSV-2 challenge.
The HSV-2 DNA copy numbers in vaginal swabs taken from mice 1, 3 and 5 days post-intravaginal challenge with 50 (1.55×104 p.f.u.) or 500×LD50 of live HSV-2 strain 186 are shown. 10 mice/group/challenge dose were used (with the exception of the positive control which used 5 mice/challenge dose). The geometric means and 95% confidence intervals are indicated. The differences between the HSV-copy numbers over days 1 to 5 for the mixed vaccine versus UD alone, W and empty vector control were significant at the 50× challenge level (*P<0.05 by the Kruskal-Wallis test and Dunn’s post test analysis of the area under the log10 copy number-time curves). This data has been published in patent US 2011/0287039 A1.
Figure 3.
HSV-2 peptide-specific T cell responses induced in mice immunized with pcDNA3-based gD2 vaccines.
2 weeks after the final immunization, splenocytes were collected and the frequency of peptide-specific IFN-γ-secreting T cells assessed by ELISPOT. Peptides 73 and 273 include known gD2 CD4 epitopes and peptides 53 and 157 include gD2 CD8 epitopes. Each point represents triplicate assays on a single mouse. Responses in empty vector immunized animals were <5 s.f.u./106 splenocytes (not shown). The average number of spots in the no peptide control wells for each vaccine was subtracted from the data prior to graphing. *** P<0.001, * P<0.05 by unpaired two-tailed t-test relative to spleen cells from mice immunized with D. Means ± SEM are indicated. The data in this figure is from 3 repeat ELISPOTs, the data from the first experiment has been published in patent US 2011/0287039 A1.
Figure 4.
Antibody responses to codon-optimized polynucleotide vaccines encoding antigen and ubiquitinated antigen.
A. Antibody titers of mice after three immunizations with 20 µg of plasmids expressing HSV-2 gD and prior to HSV-2 challenge, measured using yeast-expressed gD1 protein. B and C. Antibody titers of mice immunized as for A but not challenged, measured using yeast-expressed truncated gD1 protein and CHO cell-expressed truncated gD2 protein, respectively. Mice were immunized with pcDNA3-O-gD2 [D], a plasmid encoding a ubiquitin-tagged truncated HSV-2 gD (pcDNA3-O-Ubi-gD225–331 [UD]), a mixture of the two plasmids [mix], a codon de-optimized construct pcDNA3-W-gD2 [W], or empty pcDNA3 vector [-ve]. For parts A-C, * P<0.05, *** P<0.001, ns = not significant as measured by one-way ANOVA followed by Tukey’s Multiple Comparison test. Significance is shown relative to the UD construct. Log titers were compared so that the distributions were approximately Gaussian. n = 20, 8 and 8 for parts a, b and c, respectively. The geometric means are shown. The data in parts A and C have been published in patent US 2011/0287039 A1.
Figure 5.
Comparison of different vaccination regimes using NTC8485-O-gD2 and NTC8485-O-Ubi-gD225–331 in the HSV-2 challenge model.
A. The effect of immunization on the survival of mice vaginally challenged with 500× LD50 (1.55×105 p.f.u.) of live HSV-2 strain 186. +ve refers to the positive control mouse thymidine kinase-deficient live HSV-2 strain 333 (5×105 p.f.u./mouse); -ve refers to empty NTC8485. The survival differences between the [0.15∶0.15×2] and [0∶30 then 15∶15×2] and between the [0.15∶0.15×2] and positive control group were significant as determined by a log-rank (Mantel-Cox) test (*P<0.05). Survival rates of the active vaccine groups were all significantly higher than the negative control (P<0.05). B. IgG antibody responses to HSV gD1 on the day of challenge (day 42). Sera from mice immunized with the gD vaccines or empty vector control were assayed by ELISA. One-way ANOVA followed by Tukey’s Multiple Comparison test was used to compare vaccines (*** P<0.001, * P<0.05, ns = not significant relative to the 15∶15×3 vaccine group) as this method allows comparison of three or more unmatched groups. Log titers were compared so that the distributions were approximately Gaussian. C. Dorsal root ganglia HSV-2 DNA copy number at euthanasia in survivors of the 50× LD50 challenge 62±2 days after challenge. Each dot represents pooled ganglia from a single animal. The ratios indicate the µg of non-ubiquitinated construct: µg of ubiquitinated construct; “x2” or “x3” indicate that the vaccine was administered twice or three times, respectively. The means with SEM are shown in B and C. Ten mice/group/challenge dose were used (with the exception of the positive control which used five mice/challenge dose).
Figure 6.
HSV-2 DNA copy number in swabs from mice immunized with NTC8485-based vaccines taken after challenge.
Prior to intravaginal challenge with 50 (1.55×104 p.f.u.) or 500×LD50 of live HSV-2 strain 186, mice were immunized with the indicated vaccines. 10 mice/group/challenge dose were used (with the exception of the positive control which used 5 mice/challenge dose). Swabs were taken 1, 3 and 5 days post-challenge. The ratios indicate the µg of non-ubiquitinated construct: µg of ubiquitinated construct; “x2” or “x3” indicate that the vaccine was administered twice or three times, respectively. +ve refers to the positive control mouse thymidine kinase-deficient live HSV-2 strain 333 (5×105 p.f.u./mouse); -ve refers to empty NTC8485. The geometric means and 95% confidence intervals are shown. The vaginal swab HSV-2 copy numbers for the 15∶15×3 and the 0∶30, 15∶15×2 treatment groups (and the positive control) after challenge were all found to be significantly lower than the for the negative control group (*P<0.05, **P<0.01, ***P<0.001 by Kruskal-Wallis and Dunn’s multiple comparison test of area under the log10 copy number-day curves).