The Interaction Of Recombinant Human Mannose-binding Lectin With The Novel Influenza A (H7N9) Virus

Background:Mannose-binding lectin (MBL), a pattern-recognition molecule in serum, recognizes specific hexose sugars such as mannose and N-acetylglucosamine on bacterium, yeasts, viruses as well as apoptotic cells. It has been well-identified that MBL has antiviral effects via binding to seasonal influenza H1 and H3 subtype viruses. However, little is known about the interactions between the novel avian influenza viruses which have caused human infection recently and the innate molecules. Since February,2013, a novel reassortant H7N9 virus associated with severe human infection has emerged in eastern China, patients were presented with rapidly progressing lower respiratory tract infections and severe pneumonia or respiratory failure, with a mortality of up to 40%. The hemagglutinin (HA) gene of the novel H7N9 virus was highly homologous with that of A/duck/Zhejiang/12/2011 (H7N3) and the neuraminidase (NA) gene was closely related to that from A/wild bird/Korea/A 14/2011 (H7N9), all the internal gene segments shared the highest similarities to those from H9N2 viruses circulated in poultry in 2012. Here three viruses including the reassortants H7N9Vac and H9N2RG, which bear their corresponding HA, NA from A/Anhui/1/2013(H7N9) and A/Hongkong/33982/2009(H9N2) respectively, and internal genes of A/Puerto Rico/8/1934(H1N1)(PR8), as well as the seasonal virus A/Brisbane/I0/2007(H3N2) were tested in vitro. The interactions between recombinant human mannose-binding lectin (rhMBL) and viral particles and its interferences with virus infection were investigated. Firstly, the binding of rhMBL to viral particles was detected by the enzyme-linked immunosorbent assay (ELISA). The effects of rhMBL on the function of HA and NA were tested by hemagglutination inhibition (HI) and NA activity inhibition (NAI) assay and then the interference of rhMBL to the one-cycle and multiple-cycle infection of the influenza virus were studied. In addition, the virus labeled with Fluorescein isothiocyanate (FITC) was tested for the effects of rhMBL on viral binding and internalization to susceptible cells, Madin-Darby canine kidney cells (MDCK). The phagocytosis capabilities of human monocytes lymphoma THP1 and cytokine responses of human primary monocyte-derived macrophages (MDMs) to rhMBL-opsonized virus were also evaluated.Results:I Interaction of rhMBL with viral surface glyproteins: 1. rhMBL exhibited a strong binding to H7N9 virus as human H3N2 virus did at high virus titers, while the binding to H9N2 virus was moderate.2. rhMBL performed a significantly weaker HI activity effect on H7N9 comparing to those of H3N2 and H9N2, even at a much higher concentration (3.67±0.33 vs.0.026 ±0.001 and 0.083±0.02μg/mL, respectively).The HI activity of rhMBL on the novel H7N9 variant with an additional NGS at Residue 133 in HA emerging in 2014 showed no difference with that on wild-type.3. Minor NAI effect of rhMBL, even up to 10μg/mL, was found on H7N9 virus while it displayed significant effects on both H3N2 and H9N2 at a lowest concentration of 0.0807±0.009 and 0.0625μg/mL, respectively.4. The limited impact of rhMBL on H7N9 might result from its property of few NGS adjacent to functional region on the HA, the long distance of NGS at Residue 240, the only one located in H7 head from its corresponding RBD and the lacking of key NGS (such as Site 234 and 402) around NA activity site.Ⅱ The effects of rhMBL on viral infection and the activation of human immune cells:1. Minor neutralization activity of rhMBL on the infectivity of H7N9 virus, even up to 10μg/mL, was found. rhMBL displayed significant inhibition effects on both H3N2 and H9N2 virus at a lowest concentration of 0.053±0.02μg/mL and 0.78±0.16μg/mL, respectively.2. Minor blocking effect of rhMBL on the single-cycle infection of susceptible target cells, MDCK, with H7N9 virus at a concentration of 3μg/mL, while it significantly inhibited the entrying of H3N2 and H9N2 virus.3. rhMBL could significantly reduce the intaking of FITC-labeled H9N2 virus by MDCK cells at a concentration of 3μg/mL, while it displayed little effect on both FITC-labeled H7N9 and H3N2 virus.4. rhMBL could significantly enhance the phagocytosis of FITC-labeled H7N9 and H9N2 virus by THP1 cells at a concentration of 3μg/mL.5. H7N9 virus could induce a stronger cytokine response of human MDMs than H9N2 virus. Furthermore, both of them after being opsonized by rhMBL could trigger a much stronger releasing of IL-8, RANTES, TNF-a from human MDMs.Conclusions:1. rhMBL could bind to H7N9 virus strongly at higher virus titers.The HI, NAI effect and the neutralization activity of rhMBL, at the physiological concentrations, against H7N9 virus was limited.2. rhMBL displayed significant inhibitory effects on both H3N2 and H9N2 virus including hemagglutination, neuraminidase and neutralization activity.3. rhMBL could enhance the phagocytosis of H7N9 and H9N2 virus by THP1, also boost the productions of TNF-a, RANTES and IL-8 from human MDMs after viral infection.4. rhMBL displayed differential effects on viral infection and activation of human immune cells of the three viruses. The findings that rhMBL could not inhibit the infection of H7N9 virus while enhanced the phagocytosis and cytokine response of immune cells might in part explain the clinical severity of human H7N9 infection.