Construction of gB-null virus and of HSV-1 with gB_498-505 mutations.

<p>Line i represents the parental plasmid used for derivation of the constructs in this study, detailed previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006732#ppat.1006732.ref047" target="_blank">47</a>]. Line iii represents the replacement of the gB ORF with EGFP followed by the remaining part of the gB ORF from residue 509 to the end (gB ORF Back) that was developed to obtain a gB-null-EGFP virus. Line ii represents the HSV genome and the approximate coding position and direction of the gene for gB. Line iv represents the replacement gB genes and the site of the epitope mutations with respect to the SnaBI site used for derivation, as detailed in the text. AvrII and SnaBI are restriction sites used to clone the replacing region of gB.</p

gB-CD8⁺ T cell retention in the HSV-1 latently infected ganglia is dependent on antigen expression.

<p><b>(A)</b> Representation of infection models in which mice received unilateral corneal infections with WT (WT only) or S1L (S1L only) HSV-1, bilateral infections with WT on one cornea and S1L on the other cornea (dual infection); or bilateral corneal infection with S1L and flank infection with WT HSV-1. All corneal infections were with 1x10⁵ PFU/scarified cornea, and flank infections were with 1x10⁶ PFU on a scarified flank. At 8 or 30 dpi, TG and spleen suspensions were analyzed by flow cytometry for CD45, CD3, CD8, and gB-tetramer. <b>(B)</b> Total number of gB-tetramer⁺ cells/spleen. <b>(C and D)</b> Frequency of CD3⁺CD8⁺ T cells in the TG that are gB-tetramer⁺. * Statistical significance by one-way ANOVA with p<0.01.</p

Stimulation of acute TG-resident CD8⁺ T cell populations with WT, SIL, or L8A gB peptides.

<p>B6 mice received corneal infections with HSV-1 expressing WT, S1L, or L8A gB. TG were obtained at 8 dpi, dispersed into single cell suspensions, and the endogenous CD8⁺ T cells were stimulated for 6 hours with B6WT3 fibroblasts pulsed individually with WT, S1L, or L8A gB_498-505 peptides, in the presence of brefeldin A. Cells were surface stained for CD45 and CD8, followed by an intracellular stain for IFNγ. The data are represented as the mean percentage of CD8⁺ T cells that produced IFNγ +/- SEM (n = 5 mice per group). * represents significance of p<0.0001 for each group compared to gB peptide stimulation of wild-type infected control (first column) using one-way ANOVA with Dunnett’s multiple comparisons posttest.</p

Mutant gB proteins and recognition by gB-CD8s.

<p>Untreated B6WT3 cells (mock) or cells transfected with plasmids to express WT gB or gB_498-505 epitope mutants were incubated for eighteen hours. The labeling of the mutations is as depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006732#ppat.1006732.t001" target="_blank">Table 1</a>; WT is a plasmid which went through the mutagenesis procedure without changes. Cells were harvested for expression analysis by immunoblotting using a monoclonal gB-specific antibody (lower panel) or, in parallel, transfected cells were combined with 5x10⁴ gB-CD8s from an endogenously expanded clone and stimulated for 5 h in the presence of Brefeldin A. gB-CD8s were surface stained for CD45 and CD8, permeabilized, and stained for intracellular IFNγ. The graph depicts one of two representative experiments, with the mean percent of IFNγ⁺ cells (<i>n</i> = 2/group) and standard error of the mean (SEM) for each stimulation.</p

<i>Ex vivo</i> ganglionic reactivation of WT and S1L HSV-1.

<p>Corneas of B6 mice were infected with 1x10⁵ PFU of WT or S1L HSV-1. At 34 dpi latently infected TGs were dispersed with collagenase. <b>(A)</b> The TG cells were depleted at >95% of endogenous CD8⁺ T cells and distributed to wells of a 48-well tissue culture plate (0.2 TG equivalent/well) and cultured in culture medium containing IL-2 and with or without 2 x 10⁴ gB-CD8s added per well. Culture fluid samples were removed and replaced with fresh media every two days. The presence of infectious virus in culture fluid (indicating HSV-1 reactivation) was then determined by plaque assay. <b>(B)</b> TG cells were mock depleted or depleted of 95% of endogenous CD8⁺ T cells by treatment with anti-CD8α antibody and complement, then distributed to wells of a tissue culture plate and cultured as described in <b>A</b> above. (A & B) Data plotted as total percentage of wells that reactivated (showing infectious virus in culture supernatant) at the indicated time of culture. n = 10 TG per condition. Data for each experiment are representative of one of two repeats, but experiment-to-experiment variability in reactivation rates are routinely observed.</p

Primer sequences (complementary to the coding sequence) used in PCR to generate mutations in the gB epitope (SSIEFARL) region.

<p>Primer sequences (complementary to the coding sequence) used in PCR to generate mutations in the gB epitope (SSIEFARL) region.</p

Acute CD8⁺ T cell infiltrates in the ganglia of mice after corneal infection with WT HSV-1 or recombinant HSV-1 containing gB_498-505 mutations.

<p>Corneas of mice were infected with 1x10⁵ PFU/eye of HSV-1 WT, S1L, or L8A. At 8 dpi (peak CD8⁺ T cell infiltrate), the TG, spleen, or DLN were dissociated into single cell suspensions and surface stained with antibodies to CD45, CD3, CD8 and with MHC-I gB_498-505 tetramer as detailed in Methods. Cells were analyzed by flow cytometry, and the data are presented as the mean +/- SEM (n = 5 mice, 10 TGs) of <b>(A)</b> absolute number of CD3⁺CD8⁺ T cells per TG, <b>(B)</b> the percent of gB_498-505 tetramer positive CD8⁺ T cells in each TG, or <b>(C, D)</b> the total number of gB_498-505 tetramer specific cells per spleen and local DLN. The experiment shown is representative of three additional experiments, all producing similar results. The absolute numbers of CD8⁺ T cells induced in the TG with each virus were not statistically different as shown by a one-way ANOVA followed by Tukey’s posttest (p = 0.58).</p

The CD8⁺ T cell population in S1L infected TG contract more rapidly and contain a higher frequency of active non-gB-CD8s.

<p>B6 mice received corneal infections with either WT or the S1L mutant at 1x10⁵ PFU/cornea. TGs were harvested at 8, 12, 16, 20, or 30 dpi and: <b>(A)</b> stained for CD45, CD3, and CD8, analyzed by flow cytometry, and data recorded as the mean number of CD8⁺ T cells/TG; or stimulated for 6 hrs with <b>(B)</b> HSV-1 gB-null-EGFP infected or <b>(C)</b> PRV-gB infected B6WT3 fibroblasts in the presence of Brefeldin A. The cells were then stained for surface CD45, CD3, and CD8 and for intracellular IFNγ. Data in B and C are presented as the mean ± SEM frequency of IFNγ⁺ CD8⁺ T cells in each TG as a fraction of total CD8⁺ T cells. * p<0.05, ** p<0.01, ***p<0.001 based on a t-test comparison at each time.</p

Certain subdominant HSV-1 epitopes become more functional and arise to codominance in TG during HSV-1 S1L latency.

<p>Studies were as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006732#ppat.1006732.g009" target="_blank">Fig 9</a>, except that TGs were harvested from infected mice at 30–33 dpi. <b>(A)</b> The order of epitopes is identical to those shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006732#ppat.1006732.g009" target="_blank">Fig 9</a>. For clarity, only the non-gB_498-505 responses are shown in A, but the total percentages are displayed in (B). The bars represent the total number of CD8⁺ T cells producing IFNγ⁺ in response to peptide stimulation, and error bars represent SEM. For significantly different populations, the average fold change increase in population over wild-type is shown. <b>(B)</b> Total fraction of gB_498-505 or non-gB-CD8s depicted in figure (A) that are responding to peptide stimulations. <b>(C)</b> Total fraction of the non-gB_498-505 specific CD8+ T cells depicted in figure (A) that make IFNγ after peptide stimulation. <b>(D)</b> 30 dpi TGs suspensions were stained with tetramers specific to RR1-specific subdominant CD8+ T cell populations (tetramers for RR1 982–989 and 822–829). Shown are the total number of each RR1-specific CD8+ T cell population per TG. The total number of single functional (IFNγ⁺) or multifunctional (IFNγ⁺TNFα⁺CD107a⁺) CD8⁺ T cells after stimulation with a combination RR1 982–989 and 822–829 peptides is also shown. N = 3–7 TG equivalents per group. A t-test was performed for each matched pair of responding CD8⁺ T cells, and a * denotes p<0.05.</p

Growth of HSV gB mutants <i>in vitro</i> and <i>in vivo</i>.

<p><b>(A)</b> Virus growth in the TG of B6 mice was determined at 4 days post ocular infection with 1x10⁵ PFU of either HSV-1 WT or HSV-1 containing the gB_498-505 epitope mutants detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006732#ppat.1006732.t001" target="_blank">Table 1</a>. TG were harvested and subjected to three freeze thaw cycles and infectious virus released into the supernatant was titrated on Vero cells. The graph represents the mean virus titer for each virus ± SEM of the mean (n = 5 mice). This is a representative of two separate studies with similar results. <b>(B)</b> Genome copy number determined by qPCR in the TG of mice infected with HSV-1 WT, S1L, or L8A following harvest at day 8 post ocular infection (n = 10). Values are representative of the total copies per TG. <b>(C,D)</b> Monolayer cultures of Vero cells were infected at a multiplicity of infection (MOI) of 10 PFU/cell (high MOI Growth Curve) or 0.01 PFU/cell (Low MOI Growth Curve) respectively with HSV-1 WT, S1L, or L8A. At the indicated hours post-infection, cells and supernatants were pooled, subjected to three freeze–thaw cycles and the viral titers were determined by plaque assay. The mean PFU/culture ± standard error of the means (SEM) is shown at each time.</p