Pan-HSV-2 IgG antibody levels correlate with protection against vaginal HSV-2 MS challenge in mice and guinea pigs.

a<p>Animals were immunized with each immunogen, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g003" target="_blank">Figures 3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g004" target="_blank">4A</a>.</p>b<p>Naive and immunized mice correspond to animals presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g003" target="_blank">Figure 3</a>; guinea pigs correspond to animals presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g004" target="_blank">Figure 4</a>.</p>c<p>Mean ± sem of log (pan-HSV-2 IgG) for mice correspond to x-variables in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g003" target="_blank">Figure 3C</a>, and for guinea pigs correspond to x-variables in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g004" target="_blank">Figure 4C</a>.</p>d<p>Mean ± sem of log (reduction in vaginal HSV-2 shedding) in mice was derived from the y-variables presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g003" target="_blank">Figure 3C</a>, and for guinea pigs was derived from the y-variables presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065523#pone-0065523-g004" target="_blank">Figure 4C</a>.</p>e<p>Frequency of animals that survived until Day 30 post-HSV-2 vaginal challenge.</p>f<p>Not determined.</p>*<p>p<0.05, as determined by one-way ANOVA and Tukey's post-hoc t-test comparing immunized versus naïve animals of the same species.</p>**<p>p<0.001, as determined by one-way ANOVA and Tukey's post-hoc t-test comparing immunized versus naïve animals of the same species.</p>†<p>p = 0.01, as determined by Fisher's Exact Test comparing the frequency of survival of immunized versus naïve animals of the same species.</p>††<p>p = 0.00001, as determined by Fisher's Exact Test comparing the frequency of survival of immunized versus naïve animals of the same species.</p

Pan-HSV-2 IgG levels correlate with protection against vaginal HSV-2 challenge in mice.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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.</p

Western blot analysis to screen for candidate antibody-generating proteins of the live HSV-2 0ΔNLS vaccine.

<p>Representative Western blots of (UI) uninfected Vero cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from <b>(A)</b> mock-immunized mice (naïve) or mice immunized with <b>(B)</b> gD-2 + alum/MPL adjuvant, <b>(C)</b> HSV-2 0ΔNLS (<i>ICP0</i>^-) virus, or <b>(D)</b> an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.</p

Comparison of three methods used to measure serum levels of HSV-2-specific antibodies.

a<p>Range of HSV-2 antiserum dilutions in which estimates of anti-HSV-2 antibody abundance changed in linear relation to changes in serum dilution.</p>b<p>Mean ± sem coefficient of variation of triplicate measurements for each serum dilution in the linear range of each assay. For each serum dilution considered, the coefficient of variation = 100× standard deviation ÷ mean.</p>c<p>Goodness-of-fit (r²) of observed data relative to values predicted by a regression model within the linear range. The p-value refers to the probability that the quantity measured by each assay (i.e., neutralizing titer, OD₄₀₅, or ΔMFI) did not vary as a function of HSV-2 antiserum dilution.</p

Pan-HSV-2 IgG levels correlate with protection against vaginal HSV-2 challenge in guinea pigs.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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. (<b>E</b>) 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.</p

Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

<div><p>Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 <i>ICP0</i>^- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (<i>PLoS ONE</i> 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2’s thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine’s capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.</p></div

Western blot analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum.

<p>Western blots of (UI) uninfected Vero cells or cells inoculated with 5 pfu/cell of HSV-1 KOS, a HSV-1 ΔgD virus (KOS-gD6), HSV-2 MS, or a HSV-2 ΔgD virus (HSV-2 ΔgD-BAC) incubated with 1:20,000 dilutions of serum from mice immunized with <b>(A)</b> gD-2 + alum/MPL adjuvant or <b>(B)</b> HSV-2 0ΔNLS. Red diamonds (1–9) denote the positions of viral proteins most commonly targeted by mouse IgG antibodies.</p

Cycloheximide-release analysis segregates candidate HSV-2 0ΔNLS antigens by IE, E, or L expression kinetics.

<p>(<b>A and B)</b> Western blot of Vero cells that were uninfected (UI) or were inoculated with 5 pfu per cell of HSV-2 0ΔRING or wild-type HSV-2 MS. Virus-infected cells were treated with cycloheximide (CHX) for 10 hours followed by 7 hours of treatment with actinomycin D (ActD; lanes 1 and 6); acyclovir (ACV; lanes 2 and 7); or no drug (VEH; vehicle; lanes 3 and 8). HSV-2 0ΔRING and HSV-2 MS-infected cells that were not drug-treated (lanes 4 and 9) were included as a control, and were harvested at 17 hours p.i. (<b>A</b>) Two-color analysis of HSV-2 proteins and GFP-tagged ICP0 (expressed by HSV-2 0ΔRING) labeled with 1:20,000 mouse α-0ΔNLS antiserum (red signal) and 1:5,000 rabbit α-GFP antiserum (green signal). (<b>B)</b> Grayscale representation of mouse IgG (in 0ΔNLS antiserum) binding to HSV-2 proteins.</p

Intensity of 0ΔNLS antiserum IgG binding of HSV-2 Proteins by MW: Area-Under-Curve Analysis.

<p>^a Protein molecular weight ranges analyzed for IgG binding intensity by calculating the area under the curve for each lane profile shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116091#pone.0116091.s002" target="_blank">S2 Fig.</a></p><p>^b Mean ± sem intensity of IgG binding in the indicated MW range in the HSV-2 lane of blots shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116091#pone.0116091.g002" target="_blank">Fig. 2</a>. For each measurement, percentage of “summated signal for MW range” was calculated relative to the “summated signal for the entire HSV-2 lane.” The mean ± sem values were calculated from n = 5 Western blots incubated with serum of mice immunized with the HSV-2 0ΔNLS vaccine (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116091#pone.0116091.s001" target="_blank">S1 Fig.</a>).</p><p>Intensity of 0ΔNLS antiserum IgG binding of HSV-2 Proteins by MW: Area-Under-Curve Analysis.</p

Immunoprecipitation-mass spectrometry (IP-mass spec) analysis as a tool to screen antibody specificities in HSV-2 0ΔNLS antiserum.

<p><b>(A-B)</b> IP-mass spec experiment #1. Uninfected Vero cell proteins (UI Ag) or HSV-2 MS-infected cell proteins (HSV-2 Ag) were resuspended in a NP40-based buffer containing 150 mM NaCl and were incubated with 2% naïve mouse serum or 2% mouse 0ΔNLS-antiserum for 2 hours followed by overnight incubation with Protein A/G agarose beads. <b>(A)</b> Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 Ag + mouse 0ΔNLS antiserum versus three negative-control immunoprecipitation reactions. Black arrows denote three protein species pulled down by 0ΔNLS antiserum that were not present in controls. <b>(B)</b> Identity of proteins excised from the gel (panel A), as determined by MALDI-TOF mass spectrometry. <b>(C-D).</b> IP-mass spec experiment #2. <b>(C)</b> Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 MS-infected cell proteins (HSV-2 Ag) following incubation with 1% mouse 0ΔNLS-antiserum and Protein A/G agarose beads. The entire lane of the gel was analyzed by MALDI-TOF mass spectrometry after being cut into 18 equivalent sized slices (denoted by boxes 1–18); slice-by-slice mass spectrometry identification results for the five most abundant HSV-2 proteins are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116091#pone.0116091.s003" target="_blank">S3 Fig.</a><b>(D)</b> Number of peptide matches per positively identified HSV-2 protein. A total of 14,729 peptides were identified by mass spectrometry as being derived from 19 HSV-2 proteins that met our inclusion criteria, which were that a “positive identification” should (1) contribute >1% to the total pool of positive HSV-2 peptides (i.e., >147 peptides); (2) have >30% of its peptides recovered from 3 consecutive gel slices at the protein’s expected MW (e.g., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116091#pone.0116091.s003" target="_blank">S3 Fig.</a>); (3) have >25% of its protein sequence represented were detected by the mass spectrometer, and should (4) yield 10 or more unique peptides. Seventy-two percent of the positive HSV-2 peptides in immunoprecipitates were derived from the 5 most dominant proteins identified; namely, RR-1, ICP8, VP1–2, VP5, and gB.</p