Patient-derived head and neck tumor slice cultures: a versatile tool to study oncolytic virus action.

Head and neck cancer etiology and architecture is quite diverse and complex, impeding the prediction whether a patient could respond to a particular cancer immunotherapy or combination treatment. A concomitantly arising caveat is obviously the translation from pre-clinical, cell based in vitro systems as well as syngeneic murine tumor models towards the heterogeneous architecture of the human tumor ecosystems. To bridge this gap, we have established and employed a patient-derived HNSCC (head and neck squamous cell carcinoma) slice culturing system to assess immunomodulatory effects as well as permissivity and oncolytic virus (OV) action. The heterogeneous contexture of the human tumor ecosystem including tumor cells, cancer-associated fibroblasts and immune cells was preserved in our HNSCC slice culturing approach. Importantly, the immune cell compartment remained to be functional and cytotoxic T-cells could be activated by immunostimulatory antibodies. In addition, we uncovered that a high proportion of the patient-derived HNSCC slice cultures were susceptible to the OV VSV-GP. More specifically, VSV-GP infects a broad spectrum of tumor-associated lineages including epithelial and stromal cells and can induce apoptosis. In sum, this human tumor ex vivo platform might complement pre-clinical studies to eventually propel cancer immune-related drug discovery and ease the translation to the clinics.

A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.

Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles.

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.

Introduction to RNA Vaccines.

RNA vaccines are attractive, because they exhibit characteristics of subunit vaccines and live-attenuated vectors, including flexible production and induction of both humoral and cellular immunity. While human proof-of-concept for RNA vaccines is still pending, the nascent field of RNA therapeutics has already attracted substantial industry and government funding as well as record investments of private venture capital. Most recently, the WHO acknowledged messenger RNA (mRNA) as a new therapeutic class. In this chapter, we briefly review key developments in RNA vaccines and outline the contents of this volume of Methods in Molecular Biology.

Reduction of PMWS-associated clinical signs and co-infections by vaccination against PCV2.

The effects of a single-dose recombinant Porcine circovirus type 2 (PCV2) open reading frame 2 (ORF2) subunit vaccine were studied in a post-weaning multisystemic wasting syndrome (PMWS)-affected pig herd. A total of 1519 3-week-old piglets were allocated randomly into two treatment groups and either vaccinated against PCV2 or treated with a placebo. Study animals were followed from the time of vaccination until the end of finishing. Onset of PCV2 viraemia and clinical signs of PMWS (wasting, cough, dyspnoea, pallor and lethargy) were observed when animals were approximately 9-10 weeks old. Compared to placebo-treated animals, vaccinated animals had a significantly reduced PCV2 viral load and duration of viraemia (p < 0.0001). This reduction in viraemia was not affected by the level of maternal anti-PCV2 antibodies present at the time of vaccination. During the period of viraemia (10-26 weeks of age) vaccinated animals exhibited a 53% reduction in mortality rate (p = 0.0010), a 4.84 kg higher body weight gain (p < 0.0001) and a significant reduction in clinical signs (p < or = 0.0004). Furthermore, lung samples of vaccinated animals had a considerably reduced number of co-infections with PRRSV and Mycoplasma hyorhinis than lung samples of placebo-treated animals. These data indicate that vaccination against PCV2 alone protects pigs from clinical signs and co-infections associated with PMWS.

The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex.

A field study was conducted to investigate the effect of vaccination against porcine circovirus type 2 (PCV2) in pigs suffering from porcine respiratory disease complex (PRDC). A total of 1542 pigs were allocated randomly into two treatment groups at approximately 20 days of age. Groups received either a Baculovirus-expressed recombinant PCV2 Open Reading Frame (ORF) 2 vaccine or placebo by single intramuscular injection. Median onset of PCV2 viraemia and respiratory signs occurred when animals were 18 weeks old. Vaccination reduced the mean PCV2 viral load by 55-83% (p < 0.0001) and the mean duration of viraemia by 50% (p < 0.0001). During the period of study (from 3 to 25 weeks of age) vaccinated animals exhibited a reduced mortality rate (6.63% vs. 8.67%, difference -2.04%; p = 0.1507), an improved average daily weight gain (649 g/day vs. 667 g/day; difference +18 g/day; p < 0.0001) and a reduced time to market (164.8 days vs. 170.4 days; difference -5.6 days; p < 0.0001). The effects on performance were greatest in the 8-week period between the onset of PCV2 viraemia and the end of finishing. These data demonstrate that vaccination against PCV2 alone can significantly improve the overall growth performance of pigs in a multi-factorial, late occurring disease complex such as PRDC.

Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease.

Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease N(pro), a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein E(rns), or a combination of both mutations. Two months after infection of pregnant cattle with wild-type virus or either of the single mutants, the majority of the fetuses contained virus or were aborted or found dead in the uterus. In contrast, the double mutant was not recovered from fetal tissues after a similar challenge, and no dead fetuses were found. This result was verified with a nonrelated BVDV containing similar mutations. After intrauterine challenge with wild-type virus, mutated viruses, and cytopathogenic BVDV, all viruses could be detected in fetal tissue after 5, 7, and 14 days. Type 1 interferon (IFN) could be detected in fetal serum after challenge, except with wild-type noncytopathogenic BVDV. On days 7 and 14 after challenge, the largest quantities of IFN in fetal serum were induced by the N(pro) and RNase-negative double mutant virus. The longer duration of fetal infection with the double mutant resulted in abortion. Therefore, for the first time, we have demonstrated the essential role of both N(pro) and E(rns) RNase in blocking interferon induction and establishing persistent infection by a pestivirus in the natural host.

A glycoprotein M-deleted equid herpesvirus 4 is severely impaired in virus egress and cell-to-cell spread.

To analyse the function of the equid herpesvirus 4 (EHV-4) glycoprotein M homologue (gM), two different mutated viruses (E4DeltagM-GFP and E4DeltagM-w) were generated. Both gM-negative EHV-4-mutants were characterized on complementing and on non-complementing cells and compared with E4RgM, a virus where gM-expression had been repaired. It was demonstrated in virus growth kinetics that deleting gM had a more dramatic influence on EHV-4 replication than expected. Extracellular infectivity was detected 9-12 h later than in EHV-4-infected Vero cells and titres were reduced up to 2000-fold. In addition, mean maximal diameters of plaques were less than 20 % of diameters of wild-type plaques. These results are in contrast to most other alphaherpesviruses, including the closely related equid herpesvirus type 1, where deletion of gM only marginally influences the ability of viruses to replicate in cell culture. Nevertheless, analysis of infected cells by electron microscopy did not reveal a specific defect for deleting gM. It was concluded that EHV-4 gM is important for more than one step in virus replication in cell culture, influencing both efficient virus egress and cell-to-cell spread.

The live attenuated bovine viral diarrhea virus components of a multi-valent vaccine confer protection against fetal infection.

Fetal infection with bovine virus diarrhea virus (BVDV) causes severe economic loss and virus spread in cattle. This study investigated the ability of modified live BVDV I and II components of a commercially available modified live virus (MLV) vaccine (Breed-Back FP 10, Boehringer Ingelheim Vetmedica Inc.) to prevent fetal infection and abortion, and therefore the birth of persistently infected animals. Heifers immunized with vaccine 4-8 weeks before insemination showed no adverse effects. All vaccinated animals had seroconverted to BVDV 4 weeks after immunization. Pregnant heifers were divided into two vaccination and two control groups and challenged with type I or II BVDV on days 60-90 of gestation. Seroconversion, clinical signs, immunosuppression, viremia, mortality, abortion rate, and fetal infection were studied. Post-challenge, 6/11 (type I challenged) and 8/11 (type II challenged) vaccinated heifers were free from clinical signs of BVD. Post-challenge clinical signs noted in the vaccinated groups were mild to moderate, while all unvaccinated controls had clinical signs ranging from moderate to severe. Viremia was not detected post-challenge in any of the vaccinated heifers. However, 100% of the controls were BVDV viremic on at least 1 day post-challenge. One of 22 vaccinated heifers had transient leukopenia, whereas 2/8 and 6/7 unvaccinated heifers in control groups I and II, respectively, had transient leukopenia. Type II BVDV infection led to abortion or death in 86% of unvaccinated heifers. The corresponding vaccinated group showed no deaths or abortions. All control group fetuses were infected with BVDV. The test vaccine gave 91% (type I BVDV challenged) and 100% (type II BVDV challenged) protection from fetal infection. This vaccine is safe and effective against fetal infection, abortion (type II BVDV) and the birth of persistently infected animals.

Recovery of virulent and RNase-negative attenuated type 2 bovine viral diarrhea viruses from infectious cDNA clones.

Cloned cDNA derived from the genome of the virulent type 2 bovine viral diarrhea virus (BVDV) strain NY'93/C was sequenced and served for establishment of the infectious cDNA clone pKANE40A. Virus recovered from pKANE40A exhibited growth characteristics similar to those of wild-type BVDV NY'93/C and proved to be clinically indistinguishable from the wild-type virus in animal experiments. A virus mutant in which the RNase residing in the viral glycoprotein E(rns) was inactivated, revealed an attenuated phenotype. The plasmid pKANE40A represents the first infectious cDNA clone established for a type 2 BVDV and offers a variety of new approaches to analyze the mechanisms of BVDV-induced disease in cattle.