Investigation of multiple FDA-approved drugs against influenza infection, including an unusual A/H3N2 strain with reduced susceptibility to neuraminidase inhibitors

Abstract

In 2017, H3N2 epidemic outbreak of seasonal influenza claimed more than 400 lives in Hong Kong. Despite the unusually high mortality rate, Asn121Lys, Thr135Lys, and Ser144Lys mutations were only discovered in hemagglutinin (HA) so far, and the efficacy of vaccine was low, which could not convincingly explain why this influenza season was much more severe than that of the previous year. In this thesis, we aim to find out why the 2017 influenza season was so severe. We also tried to find a new treatment regimen for influenza infection.

In the first study (Chapter 3), we discovered that the A/Hong Kong/3391/2017 (H3N2, 3391/2017) showed a reduction in neuraminidase inhibitors (NAIs) susceptibility when compared with the 2014 strain [A/Hong Kong/485197/2014 (H3N2, 485197/2014)] in plaque reduction assay (PRA). Further analysis revealed changes in HA1 and HA2 in the 2017 strain that might have caused the low anti-viral susceptibility. In contrast to the 485197/2014, the interaction between the HA1 of 3391/2017 and its receptor could not be blocked by zanamivir. Furthermore, the HA2 of 3391/2017 had exhibited higher hemolytic activity than 485197/2014. These two changes prompted 3391/2017 to transmit faster than 485197/2014 in Madin-Darby Canine Kidney (MDCK) cells after blocking NA activity. Besides, the 3391/2017 showed more acid stability than 485197/2014.

In the second study (Chapter 4), to resolve the low susceptibility of 3391/2017 to NAIs, we explored the vitamin C (VC) in combination with NAIs in influenza treatment. In vitro, VC enhanced the efficacy of NAIs against 3391/2017 and NAI-sensitive H3N2 strains [485197/2014 and A/Hong Kong/1/1968 mouse-adapted strain(1/1968 Md)]. Moreover, antiviral-sensitive H1N1 [A/Hong Kong/415742/2009 (415742/2009)] and oseltamivir-resistant H1N1 [A/Hong Kong/423432/2009 (423432/2009)], and influenza B virus [B/TW/70555/2005 (70555/05) and B/PHU KEI/J073/2013 (J073/2013)] were more susceptible to VC-NAIs combination than to NAIs monotherapy.

In the third study (Chapter 5), we studied in vivo, the VC-zanamivir combination improved the body weight and survival rate of mice with 70555/05 and 1/1968 Md infection when compared with zanamivir alone (80% versus 20%, 70555/05; 40% versus 10%, 1/1968 Md). Furthermore, the cytokines expression and virus titre in mice were reduced by the combination treatment. Moreover, analyzing the antiviral mechanism of VC revealed that VC could bind and impact the acid stability of HA.

In the fourth study (Chapter 6), besides the combination with VC, NAIs also combined with four drugs [naproxen, chloroquine, clarithromycin, and flufenamic acid (FFA)]. There was synergy between FFA and NAIs against the 415742/2009, and the addition of clarithromycin can further enhance the antiviral effect of FFA-zanamivir combination in vitro.

In the fifth study (Chapter 7), we found a non-NAI treatment regimen (eltanexor and selinexor) which can inhibit the 415742/2009 completely in vitro.

The characteristics of low drug susceptibility and high acid stability in the 2017 strain could further explain the high virulence of the H3N2, 3391/2017 strains which caused a severe influenza outbreak in 2017. Besides that, our findings on the antiviral treatment, especially the VC-NAIs combination, could provide a new method for influenza therapy and prevent the emergence of the NAI-resistant influenza virus.