Cytokine-modified VSV is attenuated for neural pathology, but is both highly immunogenic and oncolytic

Vesicular stomatitis virus (VSV), an enveloped, nonsegmented, negative-stranded RNA virus, is being tested by several laboratories as an antitumor agent. Unfortunately, viral infection of the central nervous system (CNS) has been observed by many groups following administration to tumor-bearing animals. In rodents, VSV encephalitis is characterized by weight-loss, paralysis, and high mortality. In order to provide protection from VSV infection of the CNS after therapeutic administration, we have attenuated VSV by the introduction of the gene encoding the proinflammatory cytokine interleukin (IL)-23, and designated the new virus VSV23. We hypothesize that while VSV23 is replicating within tumors, resulting in tumor destruction, the expression of IL-23 will enhance host antitumor and antiviral immune responses. In the event that the virus escapes from the tumor, the host’s immune system will be activated and the virus will be rapidly cleared from healthy tissue. Experimental VSV23 infection of the CNS is characterized by decreased viral replication, morbidity, and mortality. VSV23 is capable of stimulating the enhanced production of nitric oxide in the CNS, which is critical for elimination of VSV from infected neurons. Intraperitoneal administration of VSV23 stimulates both nonspecific natural killer cell, virus-specific cytolytic T lymphocyte and memory virus-specific proliferative T cell responses against wild-type VSV in splenocytes. Furthermore, VSV23 is able to replicate in, and induce apoptosis of tumor cells in vitro. These data indicate that VSV23 is immunogenic, attenuated and suitable for testing as an efficacious and safe oncolytic agent

Influence of an immunodominant herpes simplex virus type 1 CD8+ T cell epitope on the target hierarchy and function of subdominant CD8+ T cells.

Herpes simplex virus type 1 (HSV-1) latency in sensory ganglia such as trigeminal ganglia (TG) is associated with a persistent immune infiltrate that includes effector memory CD8+ T cells that can influence HSV-1 reactivation. In C57BL/6 mice, HSV-1 induces a highly skewed CD8+ T cell repertoire, in which half of CD8+ T cells (gB-CD8s) recognize a single epitope on glycoprotein B (gB498-505), while the remainder (non-gB-CD8s) recognize, in varying proportions, 19 subdominant epitopes on 12 viral proteins. The gB-CD8s remain functional in TG throughout latency, while non-gB-CD8s exhibit varying degrees of functional compromise. To understand how dominance hierarchies relate to CD8+ T cell function during latency, we characterized the TG-associated CD8+ T cells following corneal infection with a recombinant HSV-1 lacking the immunodominant gB498-505 epitope (S1L). S1L induced a numerically equivalent CD8+ T cell infiltrate in the TG that was HSV-specific, but lacked specificity for gB498-505. Instead, there was a general increase of non-gB-CD8s with specific subdominant epitopes arising to codominance. In a latent S1L infection, non-gB-CD8s in the TG showed a hierarchy targeting different epitopes at latency compared to at acute times, and these cells retained an increased functionality at latency. In a latent S1L infection, these non-gB-CD8s also display an equivalent ability to block HSV reactivation in ex vivo ganglionic cultures compared to TG infected with wild type HSV-1. These data indicate that loss of the immunodominant gB498-505 epitope alters the dominance hierarchy and reduces functional compromise of CD8+ T cells specific for subdominant HSV-1 epitopes during viral latency

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

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

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