1. Microbiology and Infectious Disease
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Herpes simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin and neural disease

  1. Christopher Petro
  2. Pablo A González
  3. Natalia Cheshenko
  4. Thomas Jandl
  5. Nazanin Khajoueinejad
  6. Angèle Bénard
  7. Mayami Sengupta
  8. Betsy C Herold  Is a corresponding author
  9. William R Jacobs Jr  Is a corresponding author
  1. Albert Einstein College of Medicine, United States
  2. Howard Hughes Medical Institute, Albert Einstein College of Medicine, United States
  3. Pontificia Universidad Católica de Chile, Chile
Research Article
Cite this article as: eLife 2015;4:e06054 doi: 10.7554/eLife.06054
7 figures

Figures

Characterization of the ΔgD−/− virus.

(A) Alignment of the upstream and downstream regions of gD located within the HSV-1 BamHI J fragment encoded in VD60 cells and within the genome of ΔgD−/− using LALIGN (ExPASy) (Myers and Miller, 1988). Global alignments assess end-to-end sequences and local pairwise alignments search for regions with high identity. (B) Western blots of dextran gradient-purified virus isolated 24 hr after infection of VD60 (ΔgD−/+gD−1) and Vero (ΔgD−/−) cells. Protein expression was assessed for viral glycoproteins B (gB, UL27), gC (UL44), gD (Us6), gE (Us8), gH (Us22), gL (UL1), VP5 (UL19) and VP16 (UL48).

https://doi.org/10.7554/eLife.06054.003
HSV-2 ΔgD−/+gD−1 is attenuated in severe combined immunodeficiency (SCID) mice.

(A) Survival of SCID mice inoculated with up to 107 pfu of HSV-2 ΔgD−/+gD−1 or up to 105 pfu of the parental HSV-2(G) strain either intravaginally (ivag) or subcutaneously (sc). Statistical significance was measured by log-rank Mantel–Cox test; **p < 0.01 for ΔgD and WT after ivag inoculation. (B) Epithelial and (C) Neurological disease scores for SCID mice inoculated with the different viruses at indicated doses. (D) HSV-2 DNA (qPCR, UL30 gene) in genital tract and neural tissue samples at day 5 post-virus inoculation. The Ct cut off was determined with HSV-uninfected naïve samples. Statistical significance was measured by two-way ANOVA with Sidak's multiple comparisons test for (B, C and D); ***p < 0.001. HSV-2 ΔgD−/+gD−1 and its parental strain are abbreviated as ΔgD and WT, respectively.

https://doi.org/10.7554/eLife.06054.004
Figure 3 with 1 supplement
Vaccination with HSV-2 ΔgD−/+gD−1 protects mice against intravaginal lethal challenge.

C57BL/6 mice were subcutaneously primed and boosted 3 weeks apart either with HSV-2 ΔgD−/+gD−1 or VD60 cell lysate (Control). 21 days after boost, mice were challenged with an LD90 of wild-type HSV-2(4674) or 10 × LD90. (A) Survival, (B) Epithelial and (C) Neurological disease scores were followed daily after challenge. (D) Viral titers in vaginal washes at days 2 and 4 after challenge (n = 10 mice pooled two per group, lines indicate means). (E) Viral titers in vaginal and neural tissue (including the dorsal root ganglia, DRG) at day 5 after challenge (n = 5, lines indicate means). (F) Ex vivo reactivation of neural tissue obtained from challenged mice (n = 5 per group). (G) HSV-2 pfu in the media of ex vivo reactivated neural tissue obtained from challenged mice (n = 5 mice per group, lines indicate means). (H) HSV-2 DNA (qPCR, US6 gene) of genital tissue and neural tissue at day 5 after challenge (n = 5, lines indicate means). (I) Survival of BALB/c mice that were primed, boosted and challenged as the C57BL/6 mice described above. (J) HSV-2 DNA (qPCR, US6 gene) in Control- and ΔgD-vaccinated BALB/c neural tissue at day 7 (n = 5, lines indicate means) and ΔgD-vaccinated BALB/c neural tissue at day 21 (n = 9, lines indicate means) after challenge. HSV-2 ΔgD−/+gD−1-vaccinated group vs Control-vaccinated group were compared by log-rank Mantel–Cox test (A, I), two-way ANOVA with Sidak's multiple comparisons test (D, E, G, H) or unpaired t-test (J); ***p < 0.001. HSV-2 ΔgD−/+gD−1 and Control are abbreviated as ΔgD and Ctrl, respectively.

https://doi.org/10.7554/eLife.06054.005
Figure 3—figure supplement 1
ΔgD−/+gD−1-vaccinated cycling mice are protected against intravaginal HSV-2 challenge.

C57BL/6 mice were treated (w/MPA) or not (w/o MPA) with medroxyprogesterone (MPA) 5 days previous to subcutaneous prime and boost 3 weeks apart either with HSV-2 ΔgD−/+gD−1gD) or VD60 cell lysate (Control). 16 days after boost, all mice were treated with MPA and 5 days later challenged intravaginally with an LD90 of wild-type HSV-2(4674) (n = 5 mice per group). Statistical significance was measured by log-rank Mantel–Cox test; ***p < 0.001, treatments vs Control.

https://doi.org/10.7554/eLife.06054.006
Figure 4 with 1 supplement
Vaccination with HSV-2 ΔgD−/+gD−1 protects mice infected with HSV-2 and HSV-1 in a skin scarification model.

Mice were subcutaneously primed and boosted 3 weeks apart either with HSV-2 ΔgD−/+gD−1, Control VD60 cell lysate or PBS. 3 weeks later, mice were depilated and challenged in the flank skin with PBS (mock), (A) 5 × 104 pfu HSV-2(4674) or (B), 1 × 107 pfu HSV-1(17). Representative images are shown. (C) Skin disease scores for HSV-2(4674)-challenged mice at days 3–11. (D) Viral titers from biopsies of skin or neural tissue obtained on day 6–7 (Control mice) and day 14 (HSV-2 ΔgD−/+gD−1-vaccinated mice) (n = 5 mice per group, lines indicate means). (E) HSV-2 DNA (qPCR, US6 gene) in skin biopsies and neural tissue of Control mice (day 6–7) and HSV-2 ΔgD−/+gD−1-vaccinated mice (day 14) challenged with virus (5 mice per group, lines indicate means). Statistical significance was measured by two-way ANOVA with Sidak's multiple comparisons test (D and E); ***p < 0.001, ΔgD−/+gD−1-vaccinated group vs control group. HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD.

https://doi.org/10.7554/eLife.06054.007
Figure 4—figure supplement 1
Vaccination with HSV-2 ΔgD−/+gD−1 protects mice infected with HSV-1 in a skin scarification model.

Mice were subcutaneously primed and boosted 3 weeks apart either with HSV-2 ΔgD−/+gD−1 or Control VD60 cell lysate. 3 weeks later, mice were depilated and challenged in the flank skin with1 × 107 pfu HSV-1(17). Skin disease scores shown for challenged mice at days 3–11. Viral titers from biopsies of skin obtained on day 7–8 (Control mice) and day 14 (HSV-2 ΔgD−/+gD−1-vaccinated mice) (n = 5 mice per group, lines indicate means). Statistical significance was measured by unpaired t-test.

https://doi.org/10.7554/eLife.06054.008
Figure 5 with 1 supplement
Serum from HSV-2 ΔgD−/+gD−1-vaccinated mice protects naïve mice against HSV-2 intravaginal and skin challenge.

Mice were subcutaneously primed and boosted 3-weeks apart either with HSV-2 ΔgD−/+gD−1 or VD60 cell lysate (Control). 21 days later, blood and spleen were collected for serum and T cell purification and transferred intraperitoneally and intravenously, respectively, into naïve wild-type C57BL/6 mice. 24 hr and 48 hr after serum and T cell transfer, respectively, mice were challenged intravaginally with LD90 of HSV-2(4674) and followed for survival (n = 5 mice per group). Serum immunoglobulins were depleted using a Protein L column (A). (B) Transferred anti-HSV-2-antibodies were assessed by ELISA in vaginal washes of recipient mice (washes pooled from five mice in three independent experiments). (C) Pooled serum from Control- or HSV-2 ΔgD−/+gD−1-vaccinated mice was transferred into naïve wild-type C57BL/6 mice. 24 hr after serum transfer, mice were depilated in the flank skin and challenged with HSV-2(4674) and followed for survival (n = 5 mice per group). (D) Viral titers in skin biopsies and neural tissue of mice receiving Control-serum (day 7) and ΔgD−/+gD−1-serum (day 14) (n = 5 mice per group). (E) HSV-2 DNA (qPCR, US6 gene) in skin biopsies and neural tissue of mice receiving Control-serum (day 7) and ΔgD−/+gD−1-serum (day 14) (n = 5 mice per group). Statistical significance was measured by log-rank Mantel–Cox test (A and C), t-test (B) and two-way ANOVA with Sidak’s multiple comparisons test (D); **p < 0.01, ***p < 0.001, treatment vs control. HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD.

https://doi.org/10.7554/eLife.06054.009
Figure 5—figure supplement 1
Serum from HSV-2 ΔgD−/+gD−1-vaccinated mice protects naïve mice against epithelial and neurological disease after HSV-2 intravaginal and skin challenge.

Serum from Control- (VD60 cell lysate), ΔgD−/+gD−1-vaccinated mice, or ΔgD−/+gD−1 serum depleted of immunoglobulins using a Protein L column was transferred intraperitoneally into naïve wild-type C57BL/6 mice. 24 hr later, mice were challenged intravaginally with LD90 of HSV-2(4674) and followed for (A) epithelial and (B) neurological disease (n = 5 mice per group). (C) Serum from Control- or HSV-2 ΔgD−/+gD−1-vaccinated mice was transferred intraperitoneally into naïve wild-type C57BL/6 mice. 24 hr later, mice were depilated and challenged in the flank skin with HSV-2(4674) and followed for epithelial disease.

https://doi.org/10.7554/eLife.06054.010
Figure 6 with 1 supplement
Vaccination with HSV-2 ΔgD−/+gD−1 induces protective mucosal antibodies targeting multiple HSV proteins with ADCC activity.

(A) Anti-HSV-2 antibodies detected by ELISA in serum samples at day 7 post-boost in mice subcutaneously primed and boosted 3-weeks apart with ΔgD−/+gD−1 or VD60 lysate (Control) (4 independent pools of serum from 5–10 mice each, results shown as means ± SD). (B) Anti-HSV-2 antibodies detected by ELISA in vaginal washes at day 7 post-boost and day 4 post–challenge with HSV-2(4674) (n = 5 mice per group, lines indicate means). (C) In vitro neutralizing activity of serum antibodies (1:5 dilution) obtained from HSV-2 ΔgD−/+gD−1- or Control-vaccinated mice against HSV-2 (left) and HSV-1 (right) (n = 5 mice per group, lines indicate means). (D) Antibody-dependent cell-mediated cytotoxicity (ADCC) using mouse splenocytes, HSV-2-infected Vero cells and serum obtained either from Control- (VD60 cell lysate) or HSV-2 ΔgD−/+gD−1-vaccinated mice conducted in the absence or presence of anti-CD16/CD32 Ab to FcγRIII and FcγRII. % ADCC is defined as the percentage of dead (Live/Dead+) target cells within HSV-2 GFPHigh positive cells. A representative dot blot is shown in the upper panel and lower panel shows results for five mice per group (lines indicate means). (E) Isotype of anti-HSV-2 serum antibodies obtained from five mice each that were either HSV-2 ΔgD−/+gD−1-vaccinated and HSV-2(4674)-challenged or Control-vaccinated and HSV-2(4674)-challenged (results shown as means ± SD). (F) Western blots of cellular lysates infected with HSV-2(4674) and probed with dilutions of sera obtained from VD60 lysate-vaccinated and then subsequently infected mice (Control-Challenged) or dilutions of sera from HSV-2 ΔgD−/+gD−1-vaccinated mice 7 days post boost (ΔgD-Vaccinated); blots are representative of five independent experiments. HSV-2 ΔgD−/+gD−1-vaccinated groups were compared to control-vaccinated mice by two-way ANOVA with Sidak's multiple comparisons test (A, B, D and E) and unpaired t-test (C); *p < 0.05; **p < 0.01, ***p < 0.001. HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD.

https://doi.org/10.7554/eLife.06054.011
Figure 6—figure supplement 1
Characterization of vaginal wash and serum antibodies.

(A) Isotypes of anti-HSV-2 antibodies in vaginal washes obtained at day 4 and day 8 post intravaginal challenge in mice that were immunized with HSV-2 ΔgD−/+gD−1gD) or VD60 cell lysate (Control). Antibody responses are shown as means ± SD (n = 5 per group) and were compared by two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. (B) Western blots with sucrose gradient-purified ΔgD−/+gD−1 and ΔgD−/− viruses (equivalent particle numbers based on a Western blot for VP5) probed with an anti-gD mAb (mAb (+)), sera from HSV-2 ΔgD−/+gD−1-vaccinated mice day 7 post-boost (ΔgD) or sera from VD60 lysate-vaccinated mice day 7 post-intravaginal challenge with HSV-2(4674) (Ctrl + Challenge). (C) Western blots with purified recombinant glycoprotein B-1 (2 μg per lane) probed with an anti-gB mAb (mAb (+)), sera from VD60 lysate -vaccinated mice (Ctrl), HSV-2 ΔgD−/+gD−1-vaccinated mice day 7 post boost (ΔgD) or VD60 lysate-vaccinated mice day 7 post-intravaginal challenge with HSV-2(4674) (Ctrl + Challenge).

https://doi.org/10.7554/eLife.06054.012
Figure 7 with 1 supplement
Antibody-mediated protection requires FcγR and FcRn expression.

Survival of (A) FcγR−/− and (B) FcRn−/− mice that were either transferred serum obtained from Control- (VD60 cell lysate) or HSV-2 ΔgD−/+gD−1-vaccinated wild-type mice and then challenged intravaginally with HSV-2(4674) (n = 5 mice per group). Detection of HSV-specific Abs by ELISA in pooled vaginal washes (n = 3 pools) of (C) FcγR−/− and (D) FcRn−/− mice receiving serum (intraperitoneally) from control- (VD60 cell lysate) or HSV-2 ΔgD−/+gD−1-vaccinated wild-type mice. Survival curves were compared using log-rank Mantel–Cox test (A and B) and antibody titers using unpaired t-test (C and D); ***p < 0.001. HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD.

https://doi.org/10.7554/eLife.06054.013
Figure 7—figure supplement 1
Antibody-mediated protection requires FcγR and FcRn expression.

Epithelial and neurological disease scores in (A, B) FcγR−/− and (C, D) FcRn−/− mice receiving serum from control- or HSV-2 ΔgD−/+gD−1-vaccinated wild-type mice and then challenged intravaginally with HSV-2(4674) (n = 5 mice per group).

https://doi.org/10.7554/eLife.06054.014

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