Interfering influenza RNAs as antivirals - 'protecting virus'

Through mistakes in replication, all viruses make versions of their genomes that carry huge deletions. At the same time these small defective genomes gain the interesting property of being able to interfere intracellularly with the replication of normal virus RNAs. We thus call these interfering or protecting RNAs (iRNA) as this describes their main activities respectively in vitro and in vivo. An iRNA retains the appropropriate molecular signals and is packaged into virions that are otherwise identical with normal virions. As a result the iRNA can be delivered to the same cells that the virus infects. As a result, iRNA has the potential to protect us against infection. However, while these properties can be easily demonstrated in cell culture, there are few examples of such therapy in the scientific literature.

Interfering RNAs lack nearly all virus genes, and can only be replicated by infectious helper virus. Replication takes place when there is an excess of infectious genomes over interfering genomes, while interference occurs when interfering genomes exceed infectious genomes. Eventually interfering genomes form the major species of RNA as, being smaller, more of this RNA is made in unit time.

We work with influenza A virus, the sole cause of pandemic influenza and the major cause of epidemic/seasonal influenza. Influenza has a single stranded RNA genome of about 12 000 nt. contained in 8 separate segments. Interfering RNAs comprise about 400 nt. We have done a great deal of preliminary work using natually occurring interfering virus. This protects well in the laboratory but contains many (in excess of 50) defective genomes, and we cannot define or control these RNAs.

The breakthrough is the production of protecting influenza viruses that each has a single dominant iRNA. This can be done by producing protecting virus that contains a single cloned protecting RNA, or by selecting virus that fortuitously contains a single dominant iRNA. In fact the latter has given us our most active protecting virus. Thus we have quality control via the sequence of the protecting RNA, and quantitation through specific RT-PCR.

Interfering RNAs are highly specific and only act against the type of virus that they were derived from. However interfering influenza RNAs protect against several different influenza A viruses, and are predicted to cross react with all types of influenza A virus. This makes them a very attractive proposition as antivirals for human and veterinary use, as unlike the vaccine they do not have to be tailored to fit the new virus. In addition a single dose of protecting virus provides protection for over 6 weeks.

Protecting virus is administered by intranasal drop, although a spray might serve equally well. Only a single dose is needed, and in the lab this protects for at least 6 weeks. The next step will be to test the activity of protecting virus in people. We believe that protecting virus will provide a much needed and vital addition to our defence against future pandemics.

Virus neutralization
Antibodies are one of the major lines of defence against infection, but individual antibodies may differ in how they act and in the efficiency with which they act. At one extreme are neutralizing antibodies and at the other non-neutralizing antibodies. Both bind to virus particles but only the former inactivates infectivity. Knowledge of neutralization will help us to make vaccines that stimulate the best antibodies instead of the current procedure that stimulates a random collection of useless and highly antiviral antibodies.

Steven Reading's main effort is the investigation of microantibodies. These are small peptides (15-20 amino acid residues) that are derived from the sequence of the CDRs (complementary-determining or hypervariable regions) of virus-specific antibodies and that retain virus-neutralizing activity. He is concentrating on setting up a system that will allow us to design new microantibodies that will eact with viruses and indeed any other antigen. Microantibodies offer many advantages over conventional monoclonal antibodies: they can be chemically synthesized, and this gives a defined product that is free of biological contaminants; they can be chemically modified; and they small size allow high penetrance; in fact they can be used in all areas of biomedicine where mabs are currently employed. We are currently testing new microantibodies that have neutralizing activity against HIV.
Our work has two main aims:
(i) Development of interfering influenza RNAs as antivirals for use in humans (protecting virus)

Key references:
Noble, S., McLain, L. & Dimmock, N. J. (2004). Interfering vaccine: a novel antiviral that converts a potentially virulent infection into one that is subclinical and immunizing. Vaccine 22, 3018-3025.

Mann, A., A. C. Marriott, S. Balasingam, R. Lambkin, J. S. Oxford & Dimmock, N. J. (2006). Interfering vaccine (defective interfering influenza A virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection. Vaccine 24, 4290-4296.

(ii) Design of chemically synthesized peptides (microantibodies) that neutralize virus infectivity

Key references:
Heap, C. J., Wang, Y., Pinheiro, J. T., Reading, S. A., Jennings, K. R. & Dimmock, N. J. (2005b). Analysis of a 17 amino-acid residue virus-neutralizing antibody. J Gen Virol 86, 1791-1800.

Jackson N. A. C., Levi M., Wahren B., and Dimmock N. J. (1999) Mechanism of action of a 17 amino acid microantibody specific for the V3 loop that neutralizes free HIV-1 virions. J. Gen. Virol. 80, 225-236. [Abstract]