Vaccination against a hit-and-run viral cancer

Cancers with viral aetiologies can potentially be prevented by antiviral vaccines. Therefore, it is important to understand how viral infections and cancers might be linked. Some cancers frequently carry gammaherpesvirus genomes. However, they generally express the same viral genes as non-transformed cells, and differ mainly in also carrying oncogenic host mutations. Infection, therefore, seems to play a triggering or accessory role in disease. The hit-and-run hypothesis proposes that cumulative host mutations can allow viral genomes to be lost entirely, such that cancers remaining virus-positive represent only a fraction of those to which infection contributes. This would have considerable implications for disease control. However, the hit-and-run hypothesis has so far lacked experimental support. Here, we tested it by using Cre–lox recombination to trigger transforming mutations in virus-infected cells. Thus, ‘floxed’ oncogene mice were infected with Cre recombinase-positive murid herpesvirus-4 (MuHV-4). The emerging cancers showed the expected genetic changes but, by the time of presentation, almost all lacked viral genomes. Vaccination with a non-persistent MuHV-4 mutant nonetheless conferred complete protection. Equivalent human gammaherpesvirus vaccines could therefore potentially prevent not only viral genome-positive cancers, but possibly also some cancers less suspected of a viral origin because of viral genome loss.


INTRODUCTION
The identification of viral aetiologies for hepatic (Blumberg, 1997) and cervical (Frazer, 2004) cancers has made antiviral vaccination a relatively simple and effective means of disease prevention. Human gammaherpesviruses -Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) -are also oncogenic, but the lack of single, unifying features of the associated cancers has made it unclear how directly infection and disease are linked and so what vaccination might achieve. The robust persistence of herpesviruses in immunocompetent hosts also makes vaccination a considerable challenge.
EBV transforms B cells in vitro and, in immunocompromised patients, the viral genes responsible for transformation can cause disease (Carbone et al., 2009). However, EBVinfected cancers in immunocompetent hosts tend to express the same viral genes as non-transformed cells. They differ in also carrying oncogenic host mutations; indeed, Burkitt's lymphoma is associated more strongly with c-myc translocation than with EBV infection (Thorley-Lawson & Allday, 2008). Thus, viral genes seem mostly to have triggering or accessory roles in disease, with host oncogenes being the main drivers. The hit-and-run hypothesis proposes that viral genomes initiating disease can be lost entirely to obscure a cancer's viral origin (Ambinder, 2000). Early on, viral genes are likely to be essential for cancer-cell survival (Hammerschmidt & Sugden, 2004). However, cancers accumulate vast numbers of host mutations (Pleasance et al., 2010), some of which will inevitably promote more autonomous growth. Thus, it seems inevitable that a cancer will, with time, evolve increasing independence from viral gene functions that could allow viral genome loss.
The main problem with the hit-and-run hypothesis has been a lack of experimental support. Analyses of gammaherpesvirus-induced cancers have focused on African Burkitt's lymphoma, nasopharyngeal carcinoma and Kaposi's sarcoma, because their high frequencies of viral genome retention make plausible a causal link between infection and disease. However, focusing on virus-positive cancers tells us little about genome loss, as here most presenting cancers would be virus-negative. Instead, it is necessary to track prospectively the fate of viral genomes in transformed cells. In vitro, B-cell cancers tend to maintain gammaherpesvirus genomes, whereas Kaposi's sarcoma and nasopharyngeal carcinoma tend to lose them (Ganem, 2006;Dittmer et al., 2008). In vivo, murid herpesvirus-4 (MuHV-4) infection increases the incidence of virus-negative cancers (Sunil-Chandra et al., 1994;Tarakanova et al., 2005). However, the difficulty of analysing spontaneous cancers, where the molecular changes driving transformation are almost always unknown, makes firm functional conclusions hard to draw. To ensure that the host factors contributing to cancer remained known, we used Cre-lox recombination in a well-established conditional mouse cancer model (reviewed by DuPage et al., 2009) to transform virus-infected cells, and then analysed the emerging cancers for viral genome retention.

Analysis of virus-triggered cancers
All of the cancers analysed (n.12) were histological sarcomas (Fig. 3a). In situ hybridization ( Fig. 3b) showed surprisingly little expression of the MuHV-4 tRNAs normally abundant in lytic and latent infections (Bowden et al., 1997). At most, a few positive cells were scattered around the main cancer mass. Real-time PCR (Fig. 3c) established that sarcomas contained lower copy numbers of viral genomes than latently infected spleens of the same mice.

Viral genome loss
Fresh sarcoma explants included lymphocytes, macrophages and fibroblasts (Fig. 4a, b), but only fibroblasts grew out. Thirteen of 20 explants yielded infectious virus. Viral spread soon overwhelmed these positive cultures, consistent with fibroblasts being highly permissive for MuHV-4 lytic replication. The others remained virusnegative. At 2 days post-explant, titres were low in all cultures (,1 p.f.u. per 10 4 cells), and ,5 % of fibroblasts cloned at this time (39 of 744 clones from eight mice) yielded infectious virus. Clones lacking infectious virus also lacked viral genomes by PCR ( Fig. 4c) and Southern blotting (Fig. 4d). Nevertheless, all sarcomas showed the expected patterns of Cre-induced p53 disruption and kras(G12D) expression (Fig. 5). Therefore, the vast majority of cancer cells showed genetic changes consistent with previous virus infection but, by the time of presentation, were not virus-infected.
A trivial explanation for the lack of viral genomes in transformed cells would be that Cre uptake from infectedcell debris was sufficient for transformation. However, infecting p53 flox/flox K-ras LSL-G12D/+ mice (n524) with herpes simplex virus (HSV) expressing Cre from an HCMV IE1 promoter caused no disease. Also, Cre + HSV similarly shows no spread of Cre signal in vivo (Proença et al., 2008), and Cre + MuHV-4 plaque assays on ROSA26-lacZ flox/flox fibroblasts showed no obvious spread of b-galactosidase expression to uninfected cells.
Even when virus was recovered from cancer cells, it might have come from infiltrating, non-transformed cells rather than being that responsible for the original oncogenic hit. We examined this possibility by infecting mice with a mix of Cre + and Cre 2 MuHV-4 and typing the virus recovered from sarcomas for Cre expression. Cre + MuHV-4 showed approximately 30-fold lower latent titres than Cre 2 virus, so we used an input Cre + /Cre 2 mixture of 30 : 1. Only one of 18 virus-positive sarcoma explants was Cre + by immunofluorescence. PCR and DNA sequencing of the ORF57/58 junction showed that the Cre 2 viruses were wild-type. This did not cause sarcomas (Fig. 2), so even when virus infection was observed in sarcoma explants, it appeared rarely to be that responsible for transformation.

Vaccination against virus-triggered cancers
The high efficiency of virus-triggered oncogenesis in our model suggested that vaccine-induced protection might be difficult to achieve. However, when Cre was substituted for ORF50 to make a replication-deficient Cre + MuHV-4, both i.p. and intranasal (i.n.) infections gave no disease in p53 flox/flox K-ras LSL-G12D/+ mice over 5 months (n530). This lack of disease without lytic spread suggested that vaccination might still work -for example, the cells first encountered by incoming virions might not be transformed by k-ras. We therefore immunized p53 flox/flox K-ras LSL-G12D/+ mice either i.n. or i.p. with ORF73 2 Cre 2 MuHV-4, which lacks episome maintenance and so fails to persist in vivo (Fowler et al., 2003;Moorman et al., 2003). This protected completely against Cre + virus challenge (Fig. 6).
As a further test of vaccine efficacy, we established an i.n. Cre + virus challenge model (Fig. 7). This caused a more rapid illness than i.p. infection, with weight loss and respiratory difficulties as early as 7 days post-inoculation. The lungs of infected mice became grossly enlarged, and histological examination (Fig. 7a) showed extensive cell proliferation obliterating the alveolar air spaces. p53 flox/flox K-ras LSL-G12D/+ mice infected with Cre 2 MuHV-4 and p53 flox/flox mice infected with Cre + MuHV-4 remained clinically well, so disease again reflected k-ras activation. In situ hybridization (Fig. 7b) showed viral tRNA expression in acutely infected lungs and lymphoid tissue, but not in diseased lungs. Therefore, viral genomes were again lost rapidly from the transformed cells. Vaccination i.p. with Cre 2 ORF73 2 MuHV-4 protected completely against both macroscopic and microscopic disease (Fig. 7c-e). It also protected against the milder histological changes induced by Cre + MuHV-4 in p53 flox/flox mice (Fig. 8).

DISCUSSION
A viral aetiology is rarely considered for cancers that lack viral genomes. Our data show that cells driven to proliferate by host oncogenes readily lose gammaherpesvirus genomes in vivo. Relying on viral genome detection to establish aetiology could therefore underestimate the number of cancers to which gammaherpesviruses contribute. Most analyses of human cancers have focused on examples of genome retention. The hypothesis that these viral genomes contribute to disease (Hammerschmidt & Sugden, 2004) makes sense, as there must be a growth advantage to offset any immune recognition of viral antigens. Thus, whilst EBV genes seem not to drive the growth of EBV + Burkitt's lymphoma directly (Kang et al., 2005), they may still provide important co-factors (Thorley-Lawson & Allday, 2008). However, the retention of viral genomes by some cancer types does not establish that viral genome retention is the norm. Interestingly, whilst EBV + Burkitt's lymphoma is associated strongly with immunosuppressive malaria infection, EBV 2 Burkitt's Viral genome loss lymphoma occurs later and shows no such association. Thus, in immunocompetent hosts, EBV genome loss may be required for cancers to evolve. Viral antigen recognition (Rickinson & Moss, 1997) provides a context for understanding both genome-positive and genome-negative cancers. Cells driven to proliferate by the EBV growth programme are normally killed by antiviral T cells, so EBV-driven cancers are limited to the immunocompromised. In contrast, host mutations drive non-immunogenic cell proliferation even when the viral growth programme is turned off. This creates a new balance: viral genes are now required only for accessory roles, allowing viral antigen recognition to be reduced. However, some immune control may still occur -for example, the evasion of antigen presentation by gammaherpesvirus episome-maintenance proteins (Yin et al., 2003;Bennett et al., 2005) can fail at high proliferation rates (Münz, 2004). Also, the accumulation of host mutations is unlikely to stop. If host mutations alone remain insufficient to maintain transformation, cancer cells losing viral genomes will themselves be lost; however, if host mutations become sufficient, then antiviral T cells can select for viral genome loss.
The predominance of sarcomas in our model was surprising, as MuHV-4 classically persists in B cells (Sunil-Chandra et al., 1992). However, stromal cells may also be an important site of persistence (Stewart et al., 1998;Suárez and van Dyk, 2008) -consistent with such an idea, ORF50 2 MuHV-4 genomes were well-maintained over 3 weeks in both BHK-21 and p53 2/2 K-ras LSL-G12D/+ fibroblasts (data not shown). Stromal cells may also be more sensitive than B cells to transformation by k-ras (Nicolaides et al., 1994;Janssen et al., 2005). A key point is that known viral tropisms do not necessarily predict the cell type of virus-triggered cancers. Thus, hit-and-run oncogenesis may be more relevant to rarely EBV + cancers such as gastric adenocarcinoma (Deyrup, 2008;Shah & Young, 2009) than to those of B cells. Even in transformed fibroblasts, MuHV-4 (unlike HSV) is far from uniformly lytic , and productive MuHV-4 spread is strongly constrained in vivo by host immunity. Therefore, it would seem quite feasible for a virus-positive cancer to develop in a cell type permissive for lytic replication.
There is no certain way to identify a human cancer as previously virus-positive once it becomes virus-negative, so human gammaherpesvirus disease burdens may only be revealed by vaccination. This is not necessarily straightforward: subunit vaccines have so far failed to limit gammaherpesvirus persistence (Sokal et al., 2007;Stevenson et al., 2009). However, live-attenuated vaccines can reduce MuHV-4 latent loads (Tibbetts et al., 2003;Boname et al., 2004;Fowler & Efstathiou, 2004;Rickabaugh et al., 2004). Here, we extended this protection to a highpenetrance cancer. Latency-deficient EBV and KSHV vaccines therefore deserve serious consideration. The possibility that gammaherpesviruses contribute to more cancers than simply those remaining viral genome-positive argues that such vaccines might greatly benefit human health.
Cells. For ex vivo explants, tissues were minced finely and digested with trypsin before culture. Embryonic fibroblasts were derived from 14 day embryos. All cells were grown in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum, 2 mM glutamine, 50 mM b-mercaptoethanol (Sigma), 100 U penicillin ml 21 and 100 mg streptomycin ml 21 . All media and reagents listed here except b-mercaptoethanol were from PAA Laboratories GmbH. Fig. 5. PCR detection of Cre-mediated recombination in samples from Cre + MuHV-4-infected p53 flox/flox K-ras LSL-G12D/+ mice. (a) PCR analysis of the p53 locus of two p53 flox/flox K-ras LSL-G12D/+ mice, their primary cancers and fibroblast clones derived from them. The primers amplify the floxed p53 locus only after recombination (612 bp). Identical data were obtained for a further 10 mice. Negative images of ethidium bromide-stained PCR products are shown. (b) PCR analysis of the floxed G12D k-ras cassette of the same samples. The primers amplify the cassette (550 bp) before but not after recombination. (c) Multiplex PCR analysis of the ras locus of the same samples plus additional controls. The primers amplify from the wild-type kras locus a 622 bp band, and from the floxed G12D k-ras cassette a 500 bp band before recombination and a 650 bp band after recombination. The clones lack the 500 bp band of the parental cancers because they contain no cells with unrecombined G12D k-ras. WT, p53 flox/flox G12D k-ras "/" littermate; mut, purified 500 bp band; control DNA, non-transgenic mice.
To quantify viral DNA by Southern blotting, DNA was extracted from cells (Wizard Genomic DNA purification kit; Promega), digested with PstI, electrophoresed, transferred to Hybond nylon membranes (Roche Diagnostics), then probed with a [ 32 P]dCTP random-primed 1.2 kb PstI genomic fragment corresponding to the MuHV-4 terminal repeat unit (Efstathiou et al., 1990), washed (65 uC, 0.2 % SSC, 0.1 % SDS) and exposed to X-ray film. Recombinant viruses were analysed qualitatively for genomic structure in a similar way, except that viral DNA was digested with BglII or HindIII and probed with a BglII-restricted genomic fragment (co-ordinates 75338-78717) or the HCMV IE1-Cre construct.
b-Galactosidase assay. In vitro samples were fixed in 4 % formaldehyde (30 min), then washed in PBS and incubated (3 h, 37 uC) in PBS with 0.01 % sodium deoxycholate, 0.02 % Nonidet P-40, 2 mM MgCl 2 , 4.5 mM potassium ferricyanide, 4.5 mM potassium ferrocyanide, 1 mg X-Gal ml 21 , before washing. In vivo samples were fixed in 4 % formaldehyde (18 h) then frozen in OCT medium, sectioned, washed in PBS and developed as described above before washing and mounting.
antigens using a polyclonal rabbit serum (Sunil-Chandra et al., 1992) and for Cre recombinase using a polyclonal rabbit serum (AbCam) plus goat anti-rabbit IgG pAb (Invitrogen). The cells were mounted in ProLong Gold anti-fade reagent with DAPI (Invitrogen) and imaged using an Olympus IX70 microscope plus a Retiga 2000R camera line (QImaging). (c) p53 flox/flox K-ras LSL-G12D/+ mice were vaccinated i.p. with ORF73 " Cre " MuHV-4, and 2 months later challenged i.n. with Cre + MuHV-4. Mice were killed when they showed .20 % weight loss or progressive respiratory difficulties. The vaccinated mice remained entirely well. Equivalent data were obtained in one further experiment. (d) Ex vivo p53 flox/flox K-ras LSL-G12D/+ lungs (three per group) are shown 1 month after i.n. Cre + MuHV-4, after the same challenge but vaccinated i.p. with Cre " ORF73 " MuHV-4 2 months earlier, or without infection. Equivalent results were obtained in three further experiments. (e) Lungs of p53 flox/flox K-ras LSL-G12D/+ mice were examined by haematoxylin/eosin staining 35 days post-infection with Cre + MuHV-4. The lungs of vaccinated mice were macroscopically and histologically normal. Three representative images are shown for each group. Equivalent results were obtained in two further experiments, each with five mice per group. Bar, 100 mm.
supported by the Medical Research Council (G0701185) and the Wellcome Trust (WT089111MA).