Antiviral peptide targeting influenza and parainfluenza
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Date
28/06/2014Item status
Restricted AccessEmbargo end date
31/12/2100Author
Bacon, Matthew Neil
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Abstract
Respiratory virus infections, such as those caused by influenza, parainfluenza and
respiratory syncytial virus (hRSV), continue to be a major cause of morbidity and
mortality in both the developed and developing world. Currently, the main means of
control of influenza virus infection is vaccination, which requires advanced knowledge
of the strain that will be prevalent each year. Alternative strategies involve the use of
anti-viral drugs, which function primarily as a prophylactic. Currently, there are five
main drugs available against influenza, the adamantanes (amantadine and rimantidine)
and the neuraminidase inhibitors (oseltamivir, zanamivir and peramivir). However,
major problems exist with antivirals, notably the development of drug resistance. This
means that new drugs are urgently required that also satisfy the need to intervene at
specific phases of the infection. This thesis describes the development of a peptide
with anti-influenza virus activity (Flupep), from which a library of closely related
peptides were synthesised, with the aim of optimising antiviral efficacy.
Peptides were tested in vitro using a plaque reduction assay on cultured cell lines,
Vero and MDCK for parainfluenza and influenza respectively. Two strains of
influenza and two of parainfluenza were used, covering the main subtypes that infect
humans: Influenza A, Influenza B, PIV2 and PIV3. The plaque assay involved mixing
a fixed dose of virus with dilutions of peptide and infecting the cultured cells,
followed by incubation for between 3 and 14 days. The cells were then fixed, stained
and plaques counted as a measure of viral infectivity.
Previous work had shown that Flupep both interacts with haemagglutinin and is an
antagonist of inflammatory cytokines. As a possible explanation for antiviral activity,
binding affinity of the peptide to haemagglutinin was measured utilising enzyme
linked immunosorbent assays. However, significant binding was not detected,
suggesting non-specific binding and anti-inflammatory potential are more important
routes for antiviral activity.
Peptides which demonstrated greater than 90% plaque knockdown in vitro were
evaluated in vivo. Anaesthetised mice were infected with influenza A and administered
with the peptide concurrently. Following infection, body weights were measured daily
and clinical signs, such as shortness of breath, quality of coat and posture, were
monitored as indicators of overall health. Most mice were culled on the seventh day
post-infection and lung viral titres were determined using a plaque assay. Two
peptides were identified with high efficacy against influenza. These peptides, when
used in vivo, improved clinical signs of and dramatically reduced levels of infectious
virus in the lungs by 7 days post infection.
The peptide with highest efficacy was PEGylated and subsequently shown to possess
therapeutic potential. Intranasal administration of the PEG-peptide to anaesthetised
mice, on the two days subsequent to infection with influenza A, revealed a 17-fold fall
in lung viral titres by the fourth day post-infection. Overall, Flupep demonstrates great
potential as a future therapeutic agent for treatment of Influenza and potentially
Parainfluenza.