Probes for INS

The pathogenesis of enteric zoster, a rare debilitating complication of reactivation of latent varicella-zoster virus (VZV) in the enteric nervous system (ENS), is largely unknown. Infection of monkeys with the closely related Varicellovirus simian varicella virus (SVV) mimics VZV disease in humans. In this study, we determined the applicability of the SVV nonhuman primate model to study Varicellovirus infection of the ENS. We confirmed VZV infection of the gut in latently infected adults and demonstrated that SVV DNA was similarly present in gut of monkeys latently infected with SVV using quantitative real-time PCR. In situ analyses showed that enteric neurons expressed SVV open reading frame (ORF) 63 RNA, but not viral nucleocapsid proteins, suggestive of latent ENS infection. During primary infection, SVV-infected T-cells were detected in gut-draining mesenteric lymph nodes and located in close vicinity to enteric nerves in the gut. Furthermore, flow cytometric analysis of blood from acutely SVV-infected monkeys demonstrated that virus-infected T-cells expressed the gut-homing receptor α4β7 integrin. Collectively, the data demonstrate that SVV infects ENS neurons during primary infection and supports the role of T-cells in virus dissemination to the gut. Because SVV reactivation can be experimentally induced, the SVV nonhuman primate model holds great potential to study the pathogenesis of enteric zoster.

Primary simian varicella virus (SVV) infection in non-human primates causes varicella, after which the virus becomes latent in ganglionic neurons and reactivates to cause zoster. The host response in ganglia during establishment of latency is ill-defined. Ganglia from five African green monkeys (AGMs) obtained at 9, 13, and 20 days post-intratracheal SVV inoculation (dpi) were analyzed by ex vivo flow cytometry, immunohistochemistry, and in situ hybridization. Ganglia at 13 and 20 dpi exhibited mild inflammation. Immune infiltrates consisted mostly of CD8dim and CD8bright memory T cells, some of which expressed granzyme B, and fewer CD11c+ and CD68+ cells. Chemoattractant CXCL10 transcripts were expressed in neurons and infiltrating inflammatory cells but did not co-localize with SVV open reading frame 63 (ORF63) RNA expression. Satellite glial cells expressed increased levels of activation markers CD68 and MHC class II at 13 and 20 dpi compared to those at 9 dpi. Overall, local immune responses emerged as viral DNA load in ganglia declined, suggesting that intra-ganglionic immunity contributes to restricting SVV replication.

Human papillomavirus (HPV) infection is associated with several genetic alterations including oncogene amplification, leading to increased aggression of tumors. Recently, a relationship between HPV infection and oncogene amplification has been reported, but this finding remains controversial. This study therefore investigated relationships between HPV infection and amplification of genes in the epidermal growth factor receptor (EGFR) signaling cascade in oral squamous cell carcinoma (OSCC). Extracted DNA from 142 formalin-fixed paraffin-embedded (FFPE) OSCC tissues was performed to investigate the copy number of EGFR, KRAS, c-myc and cyclin D1 genes using real-time polymerase chain reaction (RT-PCR) and compared with calibrators. A tissue microarray of OSCC tissues was used for detection of c-Myc expression and HPV infection by immunohistochemistry and HPV E6/E7 RNA in situ hybridization, respectively. HPV infection was also investigated using PCR and RT-PCR. Of the 142 OSCC samples, 81 (57%) were HPV-infected cases. The most frequently amplified gene was c-myc (55.6%), followed by cyclin D1 (26.1%), EGFR (23.9%) and KRAS (19.7%). Amplification of c-myc was significantly associated with levels of its protein product. EGFR amplification was also significantly associated with amplification of genes in the signaling cascade: KRAS (50.0%), c-myc (34.2%) and cyclin D1 (46.0%). Interestingly, HPV infection was significantly associated with amplification of both EGFR (76.5%) and cyclin D1 (73.0%). Only cyclin D1 amplification was significantly associated with severity of OSCC histopathology. HPV infection may play an important synergistic role in amplification of genes in the EGFR signaling cascade, leading to increased aggression in oral malignancies.

We have previously demonstrated that transcriptionally active high-risk HPV (hr-HPV) is strongly incriminated in Barrett's dysplasia (BD) and oesophageal adenocarcinoma (OAC) using mainly fresh frozen tissue. This study aimed to identify biomarkers of active HPV infection in Barrett's metaplasia, (BM)/BD/OAC by immunohistochemical staining (IHC) of formalin-fixed paraffin embedded (FFPE) tissue for aberrations of p53 and the retinoblastoma (pRb) pathway which are targets for the viral oncoproteins, E6/E7 respectively. Prospectively, BM(n=81)/BD(n=72)/OAC(n=65) FFPE specimens were subjected to IHC staining for pRb, p16INK4A , cyclin D1 , p53 and RNA in-situ hybridization (ISH) for E6/E7 transcripts. HPV DNA was determined via PCR in fresh frozen specimens. Viral load measurement (real-time PCR) and Next Generation Sequencing of TP53 was also performed. Of 218 patients, 56 were HPV DNA positive [HPV16 (n=42), 18 (n=13), 6 (n=1)]. Viral load was low. Transcriptionally active HPV (DNA+ /RNA+ ) was only found in the dysplastic and adenocarcinoma group (n=21). The majority of HPV DNA+ /RNA+ BD/OAC were characterized by p16INK4Ahigh (14/21, 66.7%), pRblow (15/21, 71.4%) and p53low (20/21, 95%) and was significantly different to controls [combination of HPV DNA- /RNA- (n=94) and HPV DNA+ /RNA- cohorts (n=22)]. p53low had the strongest association with DNA+ /RNA+ oesophageal lesions (OR=23.5, 95% CI=2.94-187.8, p=0.0029). Seventeen HPV DNA+ /RNA+BD/OAC identified as p53low, were sequenced and all but one exhibited wild-type status. pRblow /p53low provided the best balance of strength of association (OR=8.0, 95% CI=2.6-25.0, p=0.0003) and sensitivity (71.4%)/specificity (71.6%) for DNA+ /RNA+ BD/OAC. Active HPV involvement in BD/OAC is characterized by wild-type p53 and aberrations of the retinoblastoma protein pathway.

Abstract

PURPOSE:

Clinical responses with programmed death (PD-1) receptor directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18 are attractive targets for therapeutic immunization, and offer an immune activation strategy that may be complementary to PD-1 inhibition.

EXPERIMENTAL DESIGN:

We report Phase Ib/II safety, tolerability and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL-12 encoding plasmids) delivered by electroporation with CELLECTRA^® constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457.

RESULTS:

MEDI0457 was associated with mild injection site reactions but no treatment related grade 3-5 adverse events (AEs). Eighteen of 21 evaluable patients showed elevated antigen specific T cell activity by IFNg ELISpot and persistent cellular responses surpassing 100 SFU/106 PBMC were noted out to one year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4/5 post-immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all five patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow cytometric analyses revealed induction of HPV16 specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%).

CONCLUSIONS:

These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.