Rhesus neutralizing antibody responses.

<p><b>(A)</b> Plasma neutralizing titers induced by the trivalent vaccine against HSV-1 and HSV-2 after the third immunization at week 12 and after a booster dose at week 40. <b>(B)</b> Neutralizing antibody titers induced by gC2 alone after the third immunization. Results are the mean of two animals. Pre-bleed represents serum obtained prior to the first gC2 immunization. <b>(C)</b> Mucosa neutralizing antibody titers to HSV-2 after the third immunization. Results are the mean and SEM of two animals. <b>(D)</b> Serum neutralizing antibody titer to HSV-2 in 10 human HSV-2 seropositive subjects with chronic HSV-2 infection and three HSV-2 seronegative controls. <b>(E)</b> Neutralization of sub-Saharan HSV-2 strains and a North American strain. 10⁵ to 10⁶ PFU of four HSV-2 strains from Uganda, Africa and one from North America (NA) were incubated with plasma from one mock (after the third immunization at week 12) or one trivalent-immunized animal (after the booster dose at week 40). Results represent the mean + SEM of three separate determinations. P values were calculated using mixed effects models to compare the mean responses. Repeated tests within an animal were adjusted for using a nested correlation structure. Sidak p-values were calculated to adjust for the multiple comparisons. *** indicates P<0.001.</p

Rhesus CD4⁺ T cell responses to the trivalent vaccine.

<p><b>(A-B)</b> PBMCs after the booster dose were stimulated <i>in vitro</i> with media, gC2, gD2 or gE2 subunit antigens. Results of each animal are shown separately. The intracellular cytokine staining (ICS) flow cytometry figures are shown to the right of each bar graph.</p

Rhesus ELISA antibody responses.

<p><b>(A)</b> Plasma antibody endpoint titers induced by the trivalent vaccine. <b>(B)</b> ELISA antibody titers in animals immunized with gC2 alone. Each animal in (A) and (B) is shown separately. Arrows indicate immunizations. <b>(C)</b> Vaginal mucosa IgG gC2, gD2 and gE2 ELISA titers to the trivalent vaccine. <b>(D)</b> Vaginal mucosa IgA responses to the trivalent vaccine were tested against the three antigens, gC2, gD2 and gE2 added to a single well. Mock vaccine responses in (C) and (D) were evaluated after the third immunization at week 12, while trivalent vaccine responses were assessed after the booster dose at week 40. Results in (C) and (D) are the mean and SEM of two animals in each group.</p

Histopathology and immunohistochemistry of vaginal mucosa in naïve and trivalent immunized rhesus macaques.

<p><b>(A)</b> Histopathology of a naïve animal two weeks after intravaginal infection. An intraepithelial vesicle and necrotic cells (NC) were detected. Magnification 20X. The boxed area shows a magnified view of a typical HSV intranuclear inclusion that is located in the tissue at the tip of the arrow. <b>(B)</b> Computer-generated magnification of the necrotic cells in Fig 5A. Thin arrows indicate hypotonic, clear fluid-filled cytoplasm, while thick arrows identify pyknotic nuclear degeneration. <b>(C)</b> Histopathology of the second naïve animal demonstrating mucosa infiltration of many lymphocytes and plasma cells. Magnification 40X. <b>(D)</b> Immunohistochemistry demonstrating moderately strong HSV-2 antigen staining (arrow) of vaginal biopsy tissues from a naïve animal eight days after the third HSV-2 intravaginal challenge. Magnification 20X. <b>(E)</b> Moderately strong HSV-2 antigen staining (arrow) of another naïve animal taken at the same time as Fig 5D. Magnification 40X. <b>(F)</b> Immunohistochemistry demonstrating no HSV-2 antigen staining of vaginal tissues obtained from a trivalent immunized animal eight days after the third HSV-2 intravaginal challenge. Magnification 20X. <b>(G)</b> Negative staining for HSV-2 antigen in the second animal immunized with the trivalent vaccine taken at the same time as Fig 5F. Magnification 40X.</p

Blocking cell-to-cell spread and binding to murine FcγRIII and FcγRIV receptors by prototype MAbs.

<p>(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.</p

Survival and genital lesions in immunized guinea pigs after intravaginal challenge.

<p><b>(A)</b> Survival curves. The table shows urinary retention and hind limb weakness during the first two weeks. <b>(B)</b> Days with acute genital lesions. <b>(C)</b> Days with recurrent genital lesions. Tables below (B) and (C) show the numbers used to generate the graphs. <b>(D)</b> Vaccine efficacy of gD2, trivalent, or trivalent + gD2 compared with mock. P values for (A) were calculated using Prism software by linear regression, while P values for (B-D) were calculated using Fisher’s exact test, except that severity of disease was calculated by Student’s t-test.</p

Guinea pig IgG ELISA and neutralizing antibody responses.

<p><b>(A)</b> IgG ELISA titers were measured at a 1:1000 dilution of serum. Samples shown are from the second experiment. P values were calculated using Fisher’s exact test. <b>(B)</b> Serum neutralizing titers with and without complement. Samples evaluated were obtained two weeks after the final immunization from animals in the first and second experiments. P values within groups comparing with and without complement were calculated by Student’s t-test, while comparisons between groups were done using the Mann Whitney test.</p

Lesion days based on antibodies produced to individual epitopes^*.

<p>Lesion days based on antibodies produced to individual epitopes^{<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007095#t002fn001" target="_blank">*</a>}.</p

Antibodies that block immune evasion activities.

<p><b>(A)</b> Top model: gC2 is a complement regulatory molecule. Activation of complement leads to deposition of complement component C3b on the virus or infected cell surface where gC2 binds to C3b and blocks downstream complement activation [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006141#ppat.1006141.ref031" target="_blank">31</a>]. Bottom model: Antibody produced by immunization with gC2 binds and blocks its interaction with C3b, enabling downstream activation of complement [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006141#ppat.1006141.ref016" target="_blank">16</a>]. <b>(B)</b> Blocking immune evasion domains on gC2. IgG obtained at week 12 after the third immunization of animals in the mock, gC2, or trivalent group was incubated with gC2 protein and added to C3b-coated wells. Results represent the mean + SEM of 2 IgG samples per group run 3 times. <b>(C)</b> Top model: gE2 is an IgG Fc binding molecule: An antibody produced by immunization with gD2 binds by its F(ab’)₂ domain to gD2 and by its Fc domain to gE2 that forms an IgG Fc receptor complex with glycoprotein I (gI2) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006141#ppat.1006141.ref025" target="_blank">25</a>]. Bottom model: antibody to gE2 produced by immunization binds and blocks its ability to interact with the Fc domain of antibody to gD2, enabling the Fc domain to activate complement and mediate antibody dependent cellular cytotoxicity [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006141#ppat.1006141.ref029" target="_blank">29</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006141#ppat.1006141.ref032" target="_blank">32</a>]. <b>(D)</b> Blocking immune evasion domains on gE2. IgG obtained prior to the first immunization or at week 40 after the fourth immunization in the trivalent group was incubated with gE2 and then added to wells coated with nonimmune human IgG. Results are expressed relative to the binding of gE2 when incubated with IgG obtained prior to the first immunization. The results represent the mean + SEM of one pre-immune IgG and two immune IgG samples tested three times. P values for (B) and (D) were calculated using mixed effects models as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006141#ppat.1006141.g002" target="_blank">Fig 2E</a>. *** indicates P<0.001.</p

Biosensor-based epitope binning.

<p>(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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007095#ppat.1007095.ref030" target="_blank">30</a>]. (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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007095#ppat.1007095.g001" target="_blank">1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007095#ppat.1007095.g002" target="_blank">2</a>. 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.</p