The Potential Applications of Reoviruses within Medicine
Introduction
A virus is 'a small infectious agent that can replicate only inside the living cells of an organism’. Around 5000 viruses have been described in detail, although there are millions of different types that exist (Wikipedia, 2012). They were first discovered in 1892 (Annual Reviews, n.d.) and are capable of infecting all organisms; from animals to plants, to bacteria to archea (Wikipedia, 2012). At the time when viruses were first discovered, the biochemistry of viruses was not known. Even now, the biochemistry of viruses is still a relatively unknown subject (Annual Reviews, n.d.).
The history of research into oncolytic viruses
A connection between cancer and viruses has been theorised for a long time. The use of viruses to destroy tumours, which is also known as viral oncolysis, dates back to the early 20th century. At this time the mechanism of cancer cell lysis was unknown, but advances in tumour biology, molecular biology and virology have played a critical role in advancing the field of oncology. Case reports of cancer regression after immunisation or infection with an unrelated virus began to appear at the beginning of the 20th century.The earliest reported case of viral oncolysis was in a patient with cervical cancer, where there was a spontaneous regression of the cancer following rabies vaccination (Kuruppu, D. and Tanabe, K.K., 2005).
Research on oncolytic viruses began soon after the early 20th century. In the 1920s, studies were carried out on animal models with oncolytic viruses to investigate whether the viruses could destroy tumours. Following this, tumour cell lysis by Newcastle disease virus and the influenza virus was observed. A decade later efforts to treat human cancers with oncolytic viruses, such as cervical cancer, began. At this stage of research, the main focus was on finding natural oncolytic viruses (Kuruppu, D. and Tanabe, K.K., 2005).
In the 1960s, further research was carried out on oncolytic viruses. Early complications began to appear, including occasional cases of uncontrolled infection, which resulted in significant morbidity and mortality of the viruses and the very frequent development of the immune response. Although these complications were harmless to the patient (Kuruppu, D. and Tanabe, K.K., 2005), the viruses were destroyed, which prevented them from destroying the cancer (Pond, A.R. and Manuelidis, E.E., 1964).
As research into the use of oncolytic viruses to treat cancer went on, it was realised that oncolytic viruses could only treat certain cancers (Kunin, C.M., 1964). In addition, when responses were observed, they were neither complete nor durable. For example, during a trial consisting of patients suffering from cervical cancer, tumour regression without toxicity was identified in more than half of the patients. Unfortunately, disease progression was observed in all of the patients. Following this, research into oncolytic viruses was nearly abandoned for a time.
However, research into oncolytic viruses continued, and all modern research consists of viruses that have been genetically modified. Due to advanced genetic engineering techniques, the viruses can be modified to express a number of desirable characteristics, which could enhance their oncolytic abilities. The viruses can be modified to be less susceptible to immune suppression, to specifically target particular classes of cancer cells, or to express desired cancer suppressing genes (Wikipedia, 2012).
What are reoviruses?
Reoviridae is a family of viruses, and one of the viruses that belong in this family is the respiratory enteric orphan virus (a reovirus). They replicate exclusively in the cytoplasm, where they enter a host cell via a receptor on the cell surface. Although this receptor is not known, it is thought to contain sialic acid and junctional adhesion molecules. Junctional adhesion molecules form continuous seals around cells to prevent water and solutes from passing freely through the paracellular space (Wikipedia, 2012).
Reoviruses are a type of oncolytic virus, which are non-enveloped in an icosahedron protein capsid. An icosahedron protein capsid is a capsid that has twenty identical equilateral triangular faces, thirty edges and twelve vertices. In addition to their protein capsid, their genome consists of double stranded RNA (Wikipedia, 2012), which is a polynucleotide made up of nucleotides that contain ribose (Jones, M., 2008).
Reoviruses have applications in everyday life as well as in the medical industry. The virus is quite stable, and can be used as a 'bio-indicator' for assessing water quality and wastewater disinfection, and has served as a model for understanding biology of double-stranded RNA and RNA transcription signals (Coombs, M.K., 2011). As mentioned before, reoviruses have been demonstrated to have applications in the medical industrydue to their oncolytic (cancer-killing) properties. They are thought to reproduce well in certain cancer cell lines (Wikipedia, 2012) and they have been shown to have many other uses in the medical industry.
How can reoviruses kill cancerous cells?
Two thirds of cancer cells possess a mutation that affects the Ras protein. When the Ras protein is activated, it can 'switch on' genes which are involved in cell growth, differentiation and survival. Mutations in the Ras protein can cause it to be 'switched on' all of the time, and this can cause unintended and overactive signals within a cell, which can ultimately lead to cancer. Reoviruses have been shown to replicate specifically in cells with an activated Ras protein (Cancer News from IRG Syndicate, 2006). The reason for this is that cells with an activated Ras protein inhibit an enzyme known as double stranded RNA kinase (PKR). PKR blocks protein synthesis when viral infection occurs, to prevent the virus from replicating. As the activated Ras protein inhibits PKR within the cancerous cells, the cancer cells are therefore susceptible to attack from the virus, as the virus will be able to replicate continuously inside of the cells. As previously stated, reoviruses can replicate specifically in cells with an activated Ras protein, and therefore cells which have inhibited PKR. Moreover, reoviruses do not target normal cells as PKR is not inhibited and the viruses will be killed if they attempt to replicate inside of the cells (Morris et al., 2008).
Research is still being carried out to investigate whether reoviruses could be used to treat cancer in the future. Dr. Matt Coffey stated that in research “Billions of particles of Reolysin®, derived from naturally occurring reovirus, were administered daily to cancer patients who had failed all other cancer therapies, or for whom no viable cancer treatments exist...the results demonstrated anti-tumor activity in patients with colorectal, prostate, pancreatic, bladder and non-small cell lung (NSCL) cancers.” Other research findings include tumour stabilisation in two colorectal patients. At three and six months, there was a reduction inCEA (a cancer marker) of 27% and 60% respectively (Cancer News from IRG Syndicate, 2006).
A formulation of reovirus, known as Reolysin®, has successfully completed phases I and II in clinical trials across a variety of cancer types (Wikipedia, 2012). There are many ongoing clinical trials, including a trial which involves the intravenous administration of Reolysin® for patients with metastatic colorectal cancer (Oncolytics Biotech Inc, 2012). Colorectal cancer is a cancer that either starts in the colon or in the rectum. Ninety-five percent of colorectal cancers are adenocarcinomas, which mean that they start in the cells that line the inside of the colon or the rectum (American Cancer Society, 2012). The trial, which involves the treatment of this cancer, has so far shown evidence of selective delivery of the virus to the tumour, and also the replication of the virus in seven out of ten of the patients. Interestingly, only necrotic tumour was found in two of the patients, and in one of these cases, the virus was detected in immune cells in the tumour. The word necrotic refers to something being dead. For example, necrotic tissue means dead tissue (Medicine Net, 2012). In this case, by describing a tumour as necrotic it is described as being dead.In six out of the ten patients there was no evidence of the virus in the normal liver surrounding the tumour, hence showing that the virus mainly targets the cancerous cells instead of normal cells. Even though some of the virus was found in normal liver in the other four patients, it was found very rarely. (Oncolytics Biotech, 2012).
Another clinical trial has been carried out with respect to Reolysin®. This trial is still taking place and is investigating the safety and efficacy of Reolysin® in the treatment of bone and soft tissue sarcomas metastatic to the lung. Although this trial has officially not ended, it aims to measure the responses of the tumour and the duration of the response and to overall evaluate the safety of multiple doses of Reolysin® (Clinical Trials, 2012).
Reoviruses and the immune response
As well as reoviruses having the ability to kill cancerous cells when they are inside the body, it is thought that they may also induce a long-lasting and powerful immune response that can continue to shrink tumours for weeks or months after the virus has left the body. This complimentary tumour-killing mechanism is based on the virus acting on specific immune system cells (Cancer News from IRG Syndicate, 2006).
It is thought that they could be 'educating' the immune system to recognise and kill the same cancer cells that were attacked by the virus. They could be doing this by activating dendritic cells, which are essential for the early detection of infection. The dendritic cells can activate the immune response by releasing cancer-fighting chemicals called cytokines. Another possibility is that the reoviruses could be instructing cells belonging to the adaptive immune system, which would continue to attack the tumour after the virus has left the body. It was shown that the reoviruses did not induce either productive infection (1000s of copies of the virus being produced), or death in dendritic cells (Cancer News from IRG Syndicate, 2006).
Reoviruses and cancer pain
Cancer pain can affect up to fifty percent of patients receiving active cancer treatment and up to ninety percent of patients with the advanced disease. Currently, opioid, non-opioid and adjuvant analgesics are used to relieve cancer pain, although all of these substances carry the risk of addiction and tolerance. In addition, there are further concerns with respect to non-opioid and adjuvant analgesics, as they can have side effects and are thought to cause gastrointestinal and renal toxicity (Morris et al., 2008). It is possible that reoviruses could be used to reduce cancer pain. In research, the administration of viruses resulted in diminished symptoms of direct-tumour associated pain, which occurred both with and without the actual tumour regression (Morris et al., 2008). This shows that it is the reoviruses that are reducing the pain, as they were able to have an effect on cancer cells that have an inactivatedRas protein as well as the cells with an activated Ras protein.
Reoviruses have been demonstrated to relieve cancer pain with respect to the neoplasm (Morris et al., 2008). A neoplasm is 'an abnormal tissue or tumor characterized by rapid cell growth, which can either be benign or cancerous' (Reference, n.d.). The virus can be administered by injection into or near to a solid neoplasm (Morris et al.,2008).Cancer pain can be reduced using a pharmaceutical composition comprising an effective amount of oncolytic virus, along with a second pharmaceutical composition comprising an effective amount of analgesic, which is preferably less than the amount of virus used. There are three serotypes of reovirus, serotype 1, serotype 2 and serotype 3. Serotype 3 is preferably used to treat cancer pain, and this serotype includes strain Dearing and strain Abney of the reovirus (Patents, 2008). The reoviruses can be 'masked' from the immune system in a liposome or in a micelle, in order that they can reach their target. Because reoviruses can be used to reduce cancer pain, they can reduce the amount of analgesic that a patient needs. This can therefore reduce the risk of side effects, tolerance, addiction and toxicity that using analgesics carries. Another benefit of using reoviruses to reduce cancer pain is that they can be combined with oncolytic viruses in different families such as the retroviridae family (Morris et al., 2008).
How can reoviruses sensitise cancerous cells to chemotherapy and radiotherapy?
Radiotherapy and chemotherapy are both common treatments used in fighting cancer. However, they are not always effective as cancer cells can become resistant to the treatment. Cancer cells, particularly those with activated Ras proteins, can be resistant to these treatments because they have a suppressed expression of the p53 gene. The p53 gene is responsible for detecting changes in the DNA and taking a cell through apoptosis if it finds any (Mombu, 2012). Apoptosis is programmed cell death, which is signalled by the nuclei of animal and human cells that are functioning normally. Apoptosis occurs when age or state of cell health and condition dictates (Biology Online, 2005). As cancer cells have suppressed this gene, the mutations caused by the chemotherapy and the radiotherapy no longer cause the cell to be killed (Mombu, 2012).
Reoviruses had some unexpected effects on non-activated Ras cells, by making them more susceptible to treatment with radiotherapy and chemotherapy. It is not known exactly how reoviruses are able to do this, but there are a number of theories into how this could work. One theory is that the Ras cells in the tumour are being destroyed by the reovirus, and the Ras cells are releasing a protein called interferon (Mombu, 2012,). Interferon is a protein, which is made and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites or tumour cells. It allows for the communication between cells to trigger the protective defences of the immune system that eradicate pathogens or tumours (Wikipedia 2012). This protein causes large amounts of natural killer cells (NK cells) to travel to the area (Mombu 2012). Natural killer cells are a type of lymphocyte, whichare critical to the innate immune system. They provide a rapid response to virally infected cells and respond to tumour formation, and they have the ability to recognise stressed cells in the absence of antibodies, allowing for a much faster immune reaction. They do not require activation in order to kill cells that appear as 'foreign' (Wikipedia 2012).The NK cells notice that the nearby cells are missing their p53 proteins from the major histocompatibility complex molecules (MHC molecules) and as a result, send cell signals to cause them to express these p53 proteins (Mombu 2012). MHC molecules are cell surface molecules which are encoded by a large gene family in all vertebrates. They mediate the interactions between leukocytes (white blood cells), and they determine the compatibility of donors for organ transplantation as well as one's susceptibility to an autoimmune disease via crossreacting immunization (Wikipedia 2012). As the p53 proteins are exposed due to the signals sent from the NK cells, any mutations caused by radiotherapy or chemotherapy would cause the cell to enter apoptosis and to die as a result (Mombu 2012).
Another, more simplistic theory into how reoviruses could make cancerous cells more sensitive to chemotherapy and radiation, is that the nearby Ras cells that are being destroyed by the reovirus are sending distress signals. The Ras cells could try to secure their DNA by increasing p53 expression, which would mean that if the DNA were to be altered in any way, the cell would go through apoptosis. In theory, the Ras cells would be increasing their susceptibility to attack (Mombu, 2012).
Reoviruses can be combined with chemotherapy drugs to treat cancer, and a particular combination of interest is reovirus and paclitaxel. The reason why this combination is of particular interest is because it is thought to promote cell-death signalling, which is more effective than using each agent on its own. Reoviruses can be combined with other chemotherapy drugs such as mitomycin, gemcitabine and cisplatin to treat non-small cell lung cancer (Cancer News from IRG Syndicate, 2006). There is an ongoing phase II study into the use of Reolysin® in combination with Paclitaxel and Carboplatin (two chemotherapy drugs), in order to treat patients with head and neck carcinoma. Preliminary assessment of a Phase I study being conducted in the United Kingdom which is investigating the combination of Reolysin®, carboplatin and paclitaxel has suggested that patients with head and neck carcinomas may be part of a group where this treatment combination is active. This phase II study is investigating how effective and how safe Reolysin® is in combination with carboplatin and paclitaxel in treating patients with head and neck carcinomas (Clinical Trials, 2012).
Squamous cell head and neck carcinoma is the sixth most common cancer in the world. There are about 640,000 cases of head and neck cancer each year, and an example of one of these cancers includes cancer of the larynx.The most common head and neck cancers are cancers of the oral cavity and cancers of the pharynx, with about 480,000 cases a year. Head and neck cancer it thought to be twice as common in men as it is in women, and the incidence of the cancer varies all across the world. However, the number of men who are suffering with it is decreasing, whereas the number of women suffering it is increasing. This is thought to be due to the increase in the number of women who smoke and drink alcohol (Oncology from Boehringer Ingelheim, n.d.).