Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling

Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis

Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins

Corneal Transduction by Intra-Stromal Injection of AAV Vectors In Vivo in the Mouse and Ex Vivo in Human Explants

The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea

HAdV-37 enters human corneal fibroblasts through caveolin-1 containing, membrane associated vesicles.

<p>Human corneal fibroblasts were fixed in 2 % paraformaldehyde and the fixed, dehydrated cell pellet embedded and sectioned at 70-90 nm. Specimens were stained with uranyl acetate and Sato’s lead stain, and viewed by transmission electron microscopy. <b>A</b>. Photo-electron micrograph of cell membrane demonstrates multiple flask shaped vesicles resembling caveolae (1), and the formation of a cavesome-like structures (2). <b>B</b>-<b>D</b>. After 30 min of absorption at 4°C, and 30 min incubation at 37°C, virus can be seen within similar structures (3), and in some cases within what appear to be early or late fusions of caveolae to form caveosomes (4). Immunoelectron microscopy for caveolin-1 (<b>E</b>, <b>F</b>) was performed on cells infected as above, but treated with 2.3 M sucrose in PBS for 15 min, frozen, and sectioned at -120° C at 50-80 nm thickness. After immunostaining, protein-A nanogold was visualized by electron microscopy. (<b>E</b>) Uninfected cells stained for caveolin-1 demonstrate binding of 10 nm gold particles to flask-shaped vesicles. (<b>F</b>) After virus infection, virus (arrows) can be seen within vesicles also associated with gold particles. Scale bar = 500 nm. </p

Caveolin-1 and cholesterol dependent viral entry.

<p>siRNA and scrambled (sc)RNA were generated and transfected into human corneal cells. <b>A</b>. Real-time RT-PCR for caveolin-1 mRNA confirmed successful reduction of caveolin-1 message (~95%) as compared to scRNA transfected cells (*p=.0002). <b>B</b>. Western blot showed reduced expression of caveolin-1 in caveolin-1 specific siRNA treated cells (right lane) as compared to untransfected and uninfected cells (left lane) or scRNA transfected, virus infected cells (middle lane) <b>C</b>. IL-8 mRNA expression after HAdV-D37 infection was reduced to almost 50% by caveolin-1 siRNA transfection, compared to scRNA (*p=.0001). </p

HAdV-37 uses caveolin-1 to enter human corneal cells.

<p><b>A</b>. Human corneal and A549 cells grown on slide chambers were infected with Cy3-labeled HAdV-D37 (red). Cells were then stained with caveolin-1 or LAMP1 antibodies as indicated followed by alexa-fluor488 (green) secondary antibodies. Original magnification: 63X. Insets represent similarly magnified squares from each photomicrograph. <b>B</b>. Three random frames from different experiments were chosen in masked fashion from each experiment (n=3) to quantify co-localization using Amira 5.2.2. In A549 cells, HAdV-C2 co-localized predominantly with caveolin-1, while HAdV-D37 co-localized with LAMP1 (*p=.02 and *p=.0025, respectively). In corneal cells (CC), Cy3-labeled HAdV-D37 co-localized predominantly with caveolin-1 (*p=.0068), and HAdV-C2 was not seen. Students <i>t</i> test was used for all comparisons. <b>C</b>. Human corneal cells were grown in chamber slides, Cy3-labelled HAdV-D37 and 488-Cholera Toxin B (CTXB) were added to the cells on ice and then warmed to 37°C, and incubated for 30, 60, and 90 min, prior confocal microscopy. HAdV-D37 (red) co-localized with CTXB (green) at all time points. At 30 min after warming, HAdV-D37 and CTXB co-localization was observed at the cell membrane, at 60 min in the cytoplasm, and at 90 min at the perinuclear region. </p

HAdV-D37 uses primarily caveolin-1 to enter corneal cells.

<p><b>A</b>. Endosomal purification was performed on postnuclear extracts from corneal cells at 1 hr post infection in 62%, 35% and 25% sucrose solution. Fractions were immunoblotted for caveolin-1 (Cav 1), pSrc, RAB5, and LAMP1. <b>B</b>. Src kinase assay was performed on endosomal fraction #’s 5-11. In this assay, fluorescence is inversely proportional to kinase activity. As shown, fractions 10 and 11, which previously showed increased caveolin-1, also demonstrated greater Src kinase activity. <b>C</b>. Fractions that had maximum expression of LAMP1 (#6) and caveolin-1 (#10) were then subjected to real time PCR. For both fractions, the quantity of viral DNA was reduced by MβCD pretreatment (*p<.05). Cholesterol replenishment resulted in restoration of E1A expression (*p<.05). There was no difference in E1A expression between virus infected cells and those pretreated with MβCD but replenished with cholesterol and then virus infected. The data presented is representative of four experiments each run in duplicate. </p

The role of caveolin-1 and Src kinase in HAdV entry and inflammation.

<p><b>A</b>. C57BL/6J wild type (WT) and caveolin-1 -/- mice (on the C57BL/6J background) were injected intrastromally with Cy3-labeled (red) HAdV-D37 (10⁵ tissue culture infectious doses) or buffer control, and the corneas harvested for confocal microscopy. Phalloidin (green) co-staining was used to better visualize the cells. Virus entry was apparent by 1 hr post infection in WT mice. In caveolin-1 -/- mice, virus remained mostly membrane bound in the first hour post infection and internalization appeared reduced at 24 hr compared to WT mice. <b>B</b>. Western blot analysis of the cornea from mice infected with HAdV-D37 revealed increasing pSrc at 1 and 24 hr post infection, while expression was minimal in untouched (uninfected) corneas. Caveolin-1 -/- mice did not show increased pSrc even after 24 hr infection. Actin controls are shown in the bottom panel. <b>C</b>. ELISA for CXCL1 chemokine performed on WT and caveolin-1 -/- mice corneas at 24 hr post infection with HAdV-D37. Infected caveolin-1 -/- mice corneas showed approximately 60% less CXCL1 expression as compared to infected wild type corneas (*p=.0001). Mock infected mice corneas did not produce any detectable CXCL1. <b>D</b>. Histology of PP2 or DMSO (control) pretreated corneas at 4 days post infection with HAdV-D37 or buffer control shows a reduction of keratitis with chemical inhibition of Src kinase. <b>E</b>. CXCL1 expression by ELISA of HAdV-D37 infected or mock infected corneas at 16 hr post infection pretreated with the Src kinase inhibitor PP2 (10 μM) or DMSO control demonstrates a significant reduction in chemokine expression due to Src inhibition (*p<0.05). </p

Phase 1 study of safety, tolerability and immunogenicity of the human telomerase (hTERT)-encoded DNA plasmids INO-1400 and INO-1401 with or without IL-12 DNA plasmid INO-9012 in adult patients with solid tumors

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is frequently classified as a \u27universal\u27 tumor associated antigen due to its expression in a vast number of cancers. We evaluated plasmid DNA-encoded hTERT as an immunotherapy across nine cancer types. METHODS: A phase 1 clinical trial was conducted in adult patients with no evidence of disease following definitive surgery and standard therapy, who were at high risk of relapse. Plasmid DNA encoding one of two hTERT variants (INO-1400 or INO-1401) with or without plasmid DNA encoding interleukin 12 (IL-12) (INO-9012) was delivered intramuscularly concurrent with the application of the CELLECTRA constant-current electroporation device 4 times across 12 weeks. Safety assessments and immune monitoring against native (germline, non-mutated, non-plasmid matched) hTERT antigen were performed. The largest cohort of patients enrolled had pancreatic cancer, allowing for additional targeted assessments for this tumor type. RESULTS: Of the 93 enrolled patients who received at least one dose, 88 had at least one adverse event; the majority were grade 1 or 2, related to injection site. At 18 months, 54.8% (51/93) patients were disease-free, with median disease-free survival (DFS) not reached by end of study. For patients with pancreatic cancer, the median DFS was 9 months, with 41.4% of these patients remaining disease-free at 18 months. hTERT immunotherapy induced a de novo cellular immune response or enhanced pre-existing cellular responses to native hTERT in 96% (88/92) of patients with various cancer types. Treatment with INO-1400/INO-1401±INO-9012 drove hTERT-specific IFN-γ production, generated hTERT-specific CD4+ and CD8+ T cells expressing the activation marker CD38, and induced hTERT-specific activated CD8 +CTLs as defined by cells expressing perforin and granzymes. The addition of plasmid IL-12 adjuvant elicited higher magnitudes of cellular responses including IFN-γ production, activated CD4+ and CD8+ T cells, and activated CD8+CTLs. In a subset analysis of pancreatic cancer patients, the presence of immunotherapy-induced activated CD8+ T cells expressing PD-1, granzymes and perforin correlated with survival. CONCLUSIONS: Plasmid DNA-encoded hTERT/IL-12 DNA immunotherapy was well-tolerated, immune responses were noted across all tumor types, and a specific CD8+ phenotype increased by the immunotherapy was significantly correlated with survival in patients with pancreatic cancer