| Popis: |
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. |
