Saturday, September 24, 2016

Cancer Fighting Recombinant Mumps Viruses

Mumps Virus Symptoms:
Swollen Cheeks and Jaws

            As of August 23rd, only 1786 cases of the mumps virus have been reported this year in the United States (1). Although relatively scarce in the United States, the mumps virus frequently infects human populations throughout Europe, Asia, and Africa (1).The mumps virus is a member of the Paramyxoviridae virus family, characterized by its negative-sense single-stranded RNA genetic component, helical protein coating, and lipid envelope. Measles virus is the more commonly known member of the Paramyxoviridae family. Mumps results in noticeably swollen cheeks and jaws, fevers, headaches, muscle aches, lethargy, and loss of appetite. However, the majority of individuals infected with mumps will not show any symptoms. A person infected with the worst of mumps’ symptoms will typically recover in a matter of days. In the worst of cases, the mumps virus may cause inflammation in the brain, testicles, ovaries, and tissue surrounding the spinal cord. Like the common cold, the mumps virus is highly contagious, spreading by both saliva and mucus. Overall, the mumps virus is quite wimpy and easily forgettable; however, recent findings by Arun Ammayappan, Stephen Russell, and Mark Federspiel (2016), surmise that the mumps virus may prove itself as a powerful tool in cancer therapy.
                  Right behind heart disease, cancer is the second leading cause of death in the United States. The use of viruses to cure cancer has been of recent interest in the 21st century. By rational logic, it appears counterintuitive to fight a disease, like cancer, with a pathogen that causes disease itself, like a virus. However, certain viruses, called “oncolytic viruses,” have been found to specifically infect and destroy cancer cells, while leaving normal cells intact. Viruses take advantage of the differences in the interferon responses (which strongly inhibit viral replication), between normal cells and cancer cells in order to solely kill the latter (2). Last October, the FDA approved the first ever oncolytic virus therapy for patients with advanced melanoma (3). This drug, Talimogene laherparepvec, is a genetically modified herpes virus that replicates in and destroys cancer cells producing a protein called granulocyte-macrophage, colony-stimulating factor. Although the first cancer-fighting virus to be approved by the FDA, Talimogene laherparepvec was not the first virus used to treat cancer patients.
In fact, during the 1970s and 1980’s, Dr. Asada, a Japanese physician injected Urabe mumps virus, isolated from an infected child, into cancer patients as a form of treatment. Out of the 90 patients that Dr. Asada treated with the mumps virus, the tumor sizes lessened by more than half in 37 patients, and tumor growth halted in another 42 patients (4). Since Dr. Asada’s initial trial, two more successful trials have been performed using the same strain (Urabe) of mumps virus (5). The second trial involved treating terminally-ill cancer patients of various forms with the Urabe mumps virus, and the third trial involved pre-immunizing advanced gynecologic cancer patients with the mumps virus before the complete cancer treatment. Dr. Asada’s success with the mumps virus led Ammayappan et al. to resurge the idea of the mumps virus as a weapon against cancer.
 Ammayappan et al. began their studies by cloning an aliquot of the original Urabe strain of mumps virus used by Dr. Asada in the 1970’s and 1980’s. The researchers developed a reverse genetics system for the mumps virus, which allowed the authors to manipulate the mumps virus by inserting specific genes into the virus that encode for particular proteins, yielding a “recombinant virus.” The authors inserted a GFP protein into the mumps virus (rMuV-UC-GFP), thus, illuminating the cell-infecting mumps virus under fluorescent microscopy. By quantifying the amount of fluorescence from the GFP protein, one could determine relative levels of viral infection in particular cell lines. The authors were also able to engineer other unique mumps viruses, including mumps viruses containing an intact luciferase gene (rMUV-UC-LUC), a human sodium iodide symporter (NIS) gene (rMUV-UC-NIS), and an interferon-𝛽 gene (rMUV-UC-mIFN𝛽), to study various aspects of the mumps virus’ ability to replicate and spread in-vivo (in a live organism). The researchers cultivated their recombinant mumps viruses in Vero cells, which lack an intact interferon response, allowing the viruses to replicate freely for use in cell culture and live mice experiments.
The propagated rMuV-UC-GFP recombinant virus was used to infect various mouse and human tumor cell lines to identify the infectivity of the virus in-vitro (outside living organisms). Fluorescent microscopy demonstrated that rMuV-UC-GFP robustly infected most human tumor cell lines, including human myeloma (Kas6/1), plasma cell leukemia (ARH-77), human multiple myeloma (MM1), ovarian carcinoma cells (SKOV3), adenocarcinoma alveolar basal epithelial cells (A549), and cervical cells (HeLa). In mice, the rMuV-UC-GFP infectivity was much lower among mouse tumor cell lines. The neuroblastoma (N2A) and colon cancer (CT-26-LacZ) cell lines showed the highest amount of infected cells, but significantly less infection than human tumor cell lines. Furthermore, the researchers succeeded in infecting rat tumor cell lines, C6 and RG2 glioma cells, finding significant levels of infection in the RG2 tumor cell line, more than any mouse tumor cell line. Ammayappan et al’s in-vitro studies of rMuV-UV-GFP in various tumor lines demonstrate the virus’ high levels of infectivity in human and rat tumor cell lines, but low level of infectivity in mouse tumor cells lines.
Figure 2a from Ammayappan et al. (2016): Green fluorescence indicates viral infection from rMUV-UC-GFP.
 Grey images illustrate tumor cells without the use of fluorescence to indicate infectivity.
 In order to identify factors other than the interferon response responsible for the lack of recombinant mumps virus replication in certain tumor cell lines, the researchers treated less permissive cell lines with a Janus kinase inhibitor called Ruxolitinib, which restricts the interferon response. As expected, the interferon inhibitor, Ruxolitinib, allowed the mumps virus to more robustly infect rat glioma, mouse lung carcinoma, and colon carcinoma cells; however, unexpectedly, plasmacytoma and human multiple myeloma cells still showed diminutive levels of viral infection. This lack of infection in the presence of Ruxolitinib suggests that certain tumor cells restrict mumps virus infection by other cellular mechanisms besides the interferon response.
 Finally, in order to test the oncolytic properties of the recombinant mumps viruses in-vivo (in a live organism), the recombinant viruses were used on mice implanted with human myeloma cells (KAS6/1). Human myeloma cancer cells implanted just under the skin of mice were allowed to develop into tumors for about five days before the mice were injected with one of the recombinant mumps viruses. The researchers used the original clone of the Urabe mumps virus (MuV-UC), an altered mumps virus that replicates more quickly than the original Urabe strain (rMuV-UC-L13328-GFP), and an altered mumps virus with a luciferase gene (rMuV- UC-LUC), which allows the virus to be observed under bioluminescent imaging due to the luciferase gene’s luminescent  products created during the virus’s replication. After sixty days, the MuV-UC treated mice demonstrated significant tumor suppression in all five mice subjects, and one observed a complete response, which is a complete absence of detectable tumor cells. Two mice had a complete response to the rMUV-UC-L13328-GFP isolate, and one complete response and a great extent of tumor suppression were noted in the mice injected with rMuV-UC-LUC. Using virus antigens, the researchers were able to detect mumps virus proteins in the harvested tumors after days 7 and 12, ensuring that each virus replicated well in the tumors. Altogether, the recombinant mumps viruses demonstrated the ability to suppress and degrade human myeloma tumor cells when implanted into mice, reinforcing the potential for the virus to be used in oncolytic therapy.
Overall, this initial recombinant mumps virus study by Ammayappan et al. (2016) suggests that recombinant mumps viruses kill human most tumor cells quite well, at least when implanted in mice, and the retain potential for future use in cancer therapy. Obviously, the ability of the mumps virus to serve as an oncolytic agent without untargeted side-effects in humans remains uncertain. Much more reproducible experimentation of the recombinant mumps viruses in-vivo must be performed. One major adversity of yielding reliable results from the in-vivo experiments on mice was the inability of the recombinant mumps virus to significantly infect mouse cells. Because the rat tumor cell lines demonstrated a much higher degree of susceptibility to mumps virus, rats may serve as a more realistic and reliable model for testing the results of mumps virus infection in human cancer cells versus non-cancer cells. Furthermore, the FDA’s neurovirulence safety test (2005), which measured nervous system damage in rats caused by mumps virus, predicted that rat-based mumps virus infections adequately parallel the potential neurovirulence of the mumps virus in humans (6 & 7). Other areas of further study include performing recombinant mumps virus experiments in-vitro and in-vivo with even more cancer forms, identifying other specific modes of viral defense in tumor cells besides the interferon response, and analyzing the compatibility of the recombinant mumps virus with other cancer therapies, such as radiation therapy or chemotherapy. Such further findings will either strengthen or weaken the possibility of recombinant mumps virus as an oncolytic agent, produce more efficient models for testing potential oncolytic viruses, and provide a greater understanding of the ability of similar viruses to alleviate cancer by targeting the degradation of tumor cells while leaving normal, healthy cells unscathed.

Ammayappan, A, Russell, S, Federspiel, M (2016). Recombinant mumps virus as a cancer therapeutic agent. Molecular Therapy- Oncolytics 3: 16018

1.     “Mumps Cases and Outbreaks.” CDC, August 23, 2016. https://www.cdc.gov/mumps/.
2.     Singh, PK, Doley, J, Kumar, GR, Sahoo, AP, Tiwari, AK (2012). Oncolytic viruses & their specific targeting to tumour cells. Indian J Med Res 136: 571-584
3.     Lawrence, L. “FDA Approves First Oncolytic Virus With New Melanoma Therapy.” Cancer Network. http://www.cancernetwork.com/melanoma/fda-approves-first-oncolytic-virus-new-melanoma-therapy.
4.     Okuno, Y, Asada, T, Yamanishi, K, Otsuka, T, Takahasi, M, Tanioka, T et al. (1977). [Mumps virus therapy of neoplasms (2)]. Nihan Rinsho 35: 3820-3825.
5.     Okuno, Y, Asada, T, Yamanishi, K, Takahashi, M, Tanioka, T et al. (1978). Studies on the use of mumps virus for treatment of human cancer. Biken J 21: 37-49.
6.     Rubin, SA, Pletnikov, M, Taffs, R, Snoy, PJ, Kobasa, D, Brown, EG et al. (2000). Evaluation of a neonatal rat model for prediction of mumps virus neurovirulence in humans. J Virol 74: 5382-5384.
7.     Rubin, SA, Afzal, MA, Powell, CL, Bentley, ML, Auda, GR, Taffs, RE et al. (2005). The rat-based neurovirulence safety test for the assessment of mumps virus neurovirulence in humans: an international collaborative study. J Infect Dis 191: 1123- 1128.

Figure 2 from Paper (Ammayappan et al., 2016)

Cancer Research Institute Video: “Oncolytic Virus Therapy: Dynamite Cure for Cancer Cells” https://www.youtube.com/watch?v=zwlCkVnUgWQ

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