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. 2013 Jun 6;8(6):e65523.
doi: 10.1371/journal.pone.0065523. Print 2013.

Pan-HSV-2 IgG Antibody in Vaccinated Mice and Guinea Pigs Correlates With Protection Against Herpes Simplex Virus 2

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Free PMC article

Pan-HSV-2 IgG Antibody in Vaccinated Mice and Guinea Pigs Correlates With Protection Against Herpes Simplex Virus 2

William P Halford et al. PLoS One. .
Free PMC article

Abstract

We lack a correlate of immunity to herpes simplex virus 2 (HSV-2) that may be used to differentiate whether a HSV-2 vaccine elicits robust or anemic protection against genital herpes. This gap in knowledge is often attributed to a failure to measure the correct component of the adaptive immune response to HSV-2. However, efforts to identify a correlate of immunity have focused on subunit vaccines that contain less than 3% of HSV-2's 40,000-amino-acid proteome. We were interested to determine if a correlate of immunity might be more readily identified if 1. animals were immunized with a polyvalent immunogen such as a live virus and/or 2. the magnitude of the vaccine-induced immune response was gauged in terms of the IgG antibody response to all of HSV-2's antigens (pan-HSV-2 IgG). Pre-challenge pan-HSV-2 IgG levels and protection against HSV-2 were compared in mice and/or guinea pigs immunized with a gD-2 subunit vaccine, wild-type HSV-2, or one of several attenuated HSV-2 ICP0 (-) viruses (0Δ254, 0Δ810, 0ΔRING, or 0ΔNLS). These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an ∼500-fold range. For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge. Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes.

Conflict of interest statement

Competing Interests: William Halford is a co-author on United States Patent Application Publication US2010/0226940 A1, which describes the uses of herpes simplex virus mutant ICP0 in the design of a live-attenuated HSV-2 vaccine strain. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Flow cytometry-based measurement of pan-HSV-2 IgG antibody levels.
A and B. Immunofluorescent labeling of fixed HSV-2 plaques with a 1∶6,000 dilution of (A) naïve mouse serum or (B) HSV-2 antiserum obtained from mice immunized with HSV-2 0ΔNLS . Mouse IgG binding was visualized with AlexaFluor594-labeled goat anti-mouse IgG (H+L). C and D. Two-color flow cytometric analysis of a fixed, single-cell suspension of CFSE-labeled, HSV-2-infected (HSV-2+) Vero cells mixed with uninfected (UI) Vero cells. Fixed cells were incubated with a 1∶6,000 dilution of (C) naïve mouse serum or (D) mouse HSV-2 antiserum and APC-labeled goat anti-mouse IgG, and were analyzed for CFSE (FL1) and APC (FL4) fluorescent intensity. E. Pan-HSV-2 IgG levels in the serum of n = 6 naïve mice versus n = 6 HSV-2 0ΔNLS-immunized mice, as determined by the ΔMFI between HSV-2+ and UI cells.
Figure 2
Figure 2. Pan-HSV-2 IgG levels correlate with protection against ocular HSV-2 challenge in mice.
(A) Design of vaccine-ocular HSV-2 challenge experiment in mice. Mice were initially inoculated in their right eye on Day 0 with culture medium or 105 pfu per eye of one of the five indicated viruses (n = 8 per group). Mice inoculated with HSV-2 MS were treated with acyclovir from Days 0 to 20 post-immunization to restrict viral pathogenesis. On Day 60, blood was harvested, and on Day 70, mice were challenged in the left eye with 105 pfu of wild-type HSV-2 MS. (B) Mean ± sem pan-HSV-2 IgG levels in pre-challenge serum, as determined by a flow cytometry-based assay. (C) For each mouse (one symbol per mouse), the average amount of infectious HSV-2 shed on Days 1, 2, and 3-post ocular challenge (y-axis) was plotted as a function of the pre-challenge HSV-2 IgG levels observed in the same mouse (x-axis). The solid black line represents the best-fit linear regression model, y = 3.35–0.56x, for the 48 matched datum pairs. (D) Mean ± sem of log (pan-HSV-2 IgG) in each immunization group is plotted on the x-axis versus mean ± sem ocular HSV-2 shedding on the y-axis. The solid black line represents the best-fit linear regression model, y = 3.44–0.64x, for these 6 matched averages (r2 = 0.86). Groups of immunized mice that exhibited a significant reduction in ocular HSV-2 shedding relative to naïve mice are indicated by a single asterisk (*; p<0.05) or double-asterisk (**; p<0.001), as determined by one-way ANOVA and Tukey's post-hoc t-test. (E) Survival frequency in each group is plotted as a function of the mean ± sem pan-HSV-2 IgG antibody level observed in each group. Groups of immunized mice that exhibited a significant difference in survival frequency relative to naïve mice are indicated by a single asterisk (*; p<0.05) or double-asterisk (**; p<0.0001), as determined by Fisher's Exact Test.
Figure 3
Figure 3. Pan-HSV-2 IgG levels correlate with protection against vaginal HSV-2 challenge in mice.
(A) Design of mouse vaccine-challenge experiment. Mice were immunized in their right, rear footpads on Day 0 with gD-2, GFP, culture medium (mock), HSV-2 0ΔNLS, or HSV-2 MS, as described in the Results (n = 10 per group). Mice immunized with HSV-2 MS received 1 mg/ml acyclovir in drinking water from Days 0 to 20 post-immunization to restrain the pathogenesis of a primary exposure to wild-type HSV-2. All mice were boosted in their left, rear footpads on Day 30 with an equivalent, booster immunization with the exception that MS-immunized mice did not require acyclovir during the boost. On Day 60, blood was harvested, and on Days 90 or 100, mice were challenged with 500,000 pfu per vagina of wild-type HSV-2 MS. Seven and 3 days prior to HSV-2 MS challenge, each mouse received a subcutaneous injection of 2 mg DepoProvera® (medoxyprogesterone) to render mouse vaginas susceptible to HSV-2 challenge. (B) Mean ± sem pan-HSV-2 IgG levels in pre-challenge serum, as determined by a flow cytometry-based assay. The frequency with which mice survived until Day 30 post-challenge is indicated. (C) For each mouse (one symbol per animal), the average amount of infectious HSV-2 shed on Days 1, 3, 5, and 7 post-vaginal challenge (y-axis) was plotted as a function of pre-challenge pan-HSV-2 IgG levels observed in the same mouse (x-axis). The solid black line represents the best-fit linear regression model, y = 3.85–0.76x, for the 50 matched datum pairs. (D) Mean ± sem of log (pan-HSV-2 IgG) in each immunization group is plotted on the x-axis versus mean ± sem vaginal HSV-2 shedding on the y-axis. The solid black line represents the best-fit linear regression model, y = 3.89–0.79x, for these 5 matched averages (r2 = 0.98). Groups of immunized mice that exhibited a significant reduction in vaginal HSV-2 shedding relative to naïve mice are indicated by a single asterisk (*; p<0.05) or double-asterisk (**; p<0.001), as determined by one-way ANOVA and Tukey's post-hoc t-test.
Figure 4
Figure 4. Pan-HSV-2 IgG levels correlate with protection against vaginal HSV-2 challenge in guinea pigs.
(A) Design of guinea pig vaccine-challenge experiment. Guinea pigs were immunized in their right, rear footpads on Day 0 with gD-2, culture medium (mock), HSV-2 0ΔNLS, or HSV-2 MS, as described in the Results (n = 5 per group). Guinea pigs immunized with HSV-2 MS received 1 mg/ml acyclovir in drinking water from Days 0 to 20 post-immunization to restrain the pathogenesis of a primary exposure to wild-type HSV-2. All guinea pigs were boosted in their left, rear footpads on Day 30 with an equivalent, booster immunization; MS-immunized guinea pigs did not receive acyclovir during the secondary boost. On Day 75, blood was harvested, and on Day 90, guinea pigs were challenged with 2×106 pfu per vagina of wild-type HSV-2 MS. (B) Mean ± sem pfu of HSV-2 shed per vagina between Days 1 and 8 post-challenge in guinea pigs that were naïve (n = 5) or were immunized with gD-2+ alum/MPL (n = 4), HSV-2 0ΔNLS (n = 5), or an acyclovir (ACV)-restrained HSV-2 MS infection (n = 5). A single asterisk (*) denotes p<0.05 and a double asterisk (**) denotes p<0.0001 that HSV-2 MS vaginal shedding was equivalent to naïve guinea pigs on that day, as determined by one-way ANOVA and Tukey’s post hoc t-test. (C) For each guinea pig (one symbol per animal), the average amount of infectious HSV-2 shed on Days 1, 2, 3, 4, 6, and 8 post-vaginal challenge (y-axis) was plotted as a function of pre-challenge pan-HSV-2 IgG levels observed in the same guinea pig (x-axis). The solid black line represents the best-fit linear regression model, y = 3.77–0.95x, for these 19 matched datum pairs. (D) Mean ± sem of log (pan-HSV-2 IgG) in each immunization group is plotted on the x-axis versus mean ± sem vaginal HSV-2 shedding on the y-axis. The solid black line represents the best-fit linear regression model, y = 3.77–0.95x, for these 4 matched averages (r2 = 0.98). Groups of immunized guinea pigs that exhibited a significant reduction in vaginal HSV-2 shedding relative to naïve guinea pigs are indicated by a single asterisk (*; p<0.05) or double-asterisk (**; p<0.001), as determined by one-way ANOVA and Tukey's post-hoc t-test. (E) The worst case of perivaginal disease in each group of naïve or immunized guinea pigs on Day 7 post-challenge. Survival frequency refers to the frequency with which animals in each immunization group survived until Day 30 post-challenge.
Figure 5
Figure 5. Adoptive transfer of HSV-2 antiserum provides limited protection against ocular HSV-2 MS challenge.
Female, age-matched strain 129 mice received either i. an adoptive transfer of 0.25 ml naïve serum prior to challenge; ii. an adoptive transfer of 0.25 ml HSV-2 antiserum just prior to challenge; or iii. active immunization with the live HSV-2 0ΔNLS virus 90 and 60 days prior to challenge. Mice were challenged in both eyes with 100,000 pfu per eye of HSV-2 MS, and challenge virus shedding and disease onset were recorded. (A and B) Mean ± sem of HSV-2 shedding from mouse eyes on (A) Day 1 and (B) Day 3 post-challenge (n = 5 per group). (C) Mean ± sem duration of survival of each group of mice. Red numbers over each bar report the frequency of 'survival' and 'disease incidence' in each group of mice. Significant increases in the duration of survival relative to naïve mice are indicated by a single asterisk (*; p<0.05) or double asterisk (**; p<0.001), as determined by one-way ANOVA and Tukey's post-hoc t-test.

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