Summary of the invention
Professor Nigel Dimmock and colleagues have discovered how to use protecting RNA to prevent influenza in two in vivo systems, mice and ferrets. A protecting RNA arises naturally and is incorporated into a virus particle in place of one of the full-length RNA segments to form a protecting virus. The flu genome comprises 8 unlinked segments of single stranded RNA, and Protecting RNAs have huge but specific deletions of 75-80% of internal sequence. The protecting virus particle provides a perfect delivery vehicle which allows the RNA to target the same cells as those infected by infectious virus. By definition, protecting virus is non-infectious and requires helper RNA from an infectious virus to enable it to be replicated. In the presence of infectious virus, the small protecting RNA is replicated faster than the larger wild-type RNA and hence is incorporated into progeny virus in preference to the full-length segment. This has two advantages: firstly most of the progeny virus is non-infectious, and secondly, yet more protecting RNA is delivered to neighbouring cells. Protection depends on the ratio of protecting virus/RNA: infectious virus/RNA. In animals the resulting decrease in infectious virus progeny completely prevents any sign of clinical disease, and allows the host defence mechanisms time to develop and clear the infection. A single dose of protecting virus can be given to mice at least 6 weeks before they are infected. All these mice remain well, as opposed to mice given inactivated protecting virus, which lose weight, and become very ill or die within one week. Professor Dimmock has also shown that protecting virus in ferrets – the gold standard model for human influenza – is equally effective at combatting clinical flu.
There are many different sequences of protecting RNA: they can arise from any of the 8 genomic segments, and RNAs from one segment can have different deletions. Professor Dimmock suspects that different protecting RNA sequences have different specific protecting activities.
Professor Dimmock has used reverse genetics to produce two cloned protecting viruses that each contain a single defined deleted RNA and published a paper demonstrating that these delay onset of clinical disease – i.e. these are genuine protecting RNAs. Since then he has overcome the problem of how to propagate sufficient quantities of cloned interfering virus and has produced preparations that completely prevent clinical disease in otherwise lethally infected mice. In addition he has isolated a spontaneously derived protecting virus that contains a single protecting RNA.
To date protecting virus has been shown to protect against H1N1, H2N2, H3N2 and H3N8 viruses, and is predicted to protect against all subtypes of flu A virus, as it acts through the intracellular replicative mechanism, rather than on the highly variable protein coat. This broad spectrum protection is in contrast to vaccines, which are effective only against one strain and have to be frequently revised due to virus mutation. Importantly, cloned protecting virus can be grown in embryonated chicken’s eggs like the conventional vaccine, and could be manufactured by the same production line. Animals protected from virulent infectious virus by protecting virus develop a conventional immunity to that virus, as there is low level virus replication. Thus in effect, protecting virus converts the virulent virus into a harmless live vaccine.
Protecting virus could be developed into a method of prophylaxis against flu. It is equally applicable to human and veterinary use, including the poultry and race horse industries. Current experiments demonstrate that immunity against the protecting flu itself is not immediately induced in animal models, although this arises with repeat doses. However there are 16 different HA proteins that could be incorporated into helper viruses to solve this issue. Delivery of the interfering virus is by the intranasal route – at present in the form of drops applied directly to the nostrils, but given the correct formulation a nasal spray should be possible as well. As protecting virus has to reach the cells of the respiratory tract that are susceptible to infection by wildtype flu administration by injection would not be possible, but it is not considered that this would be a disadvantage.
Professor Dimmock has completed the majority of the laboratory work in animal models that is possible at this stage. We believe that the next major milestone in the development process will be to move towards human trials of protecting flu. At this stage we do not anticipate complications in gaining ethical approval; protecting flu is based on the standard strains of flu commonly used in vaccines, and since the protecting RNAs were naturally arising we believe they should not be counted as genetically modified organisms.
- Protecting RNAs have been cloned and a technique for producing sufficient amounts of protecting virus has been developed.
- The naturally selected protecting virus currently being used in protecting virus gives up to 100-fold better protection against flu than the other cloned protecting RNAs (unpublished data).
- This protecting RNA is very active and a single dose has been shown to prevent disease from virus inoculated 6 weeks later. (Tamiflu and Relenza - the main antiviral drugs - have to be given twice daily and within 24 to 48 hours of infection).
- Protecting virus has recently been demonstrated to completely protect mice from lethal infection by influenza virus when administered up to 24 hours post infection. This was surprising as previously identified interfering viruses had no therapeutic activity (unpublished data).
- Two patent applications have been filed with the UK patent office (unpublished). These cover the sequences of the most active protecting RNA molecules, as well as the use of cloned protecting viruses as both prophylactic and therapeutic medicaments.