A non-VH1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against H1N1 and H5N1 viruses

Abstract

Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.

Figure 1. PN-SIA49 confers protection in mice against lethal challenge with both A/Wilson Smith/33 and A/Vietnam/1203/2004.

Female BALB/c mice were challenged with 3 LD50 of A/Wilson Smith/33 (WS33) or A/Vietnam/1203/2004 (VN04). Twenty-four hours after the viral challenge, graded doses (10, 1, 0.1 mg/Kg) of PN-SIA49 or the control antibody (e137, 10 mg/Kg) were administrated to mice. Mice were monitored for body weight loss and survival for 2 weeks after challenge. (A) Body weight following WS33 virus challenge and (B) VN04 and treated with PN-SIA49. (C) Survival rates of mice challenged with WS33 and (D) VN04 after PN-SIA49 treatment. (E) Viral titers in the lungs of mice challenged with WS33 and (F) VN04 on day 4 post infection. Each data point represents the average viral titer from 4 mice. A group of mice treated with a negative control mAb (e137) was used and is included in each panel for direct comparison.

Figure 2. PN-SIA49 protects H1-HA from the low pH induced protease sensitivity.

Western Blotting results of the protease susceptibility assay for H1-HA. Exposure to low pH converts HA to the protease-susceptible, post-fusion state (lane 6 and 7). Pre-treatment with PN-SIA49 partially blocks the pH-induced conformational change retaining HA in the protease-resistant, pre-fusion state (lane 3 and 4). In lanes 2–4 is also visible the signal given by PN-SIA49 (180 KDa). The other bands (about 110 KDa and 190 KDa) are due to HA aggregates bound by PN-SIA49 or by the primary anti-influenza Ab used in the western blotting assay.

Figure 3. Amino acidic sequence conservation in hemagglutinin groups and subtypes at the region bound by PN-SIA49.

Circles below residues indicate PN-SIA49 percentage binding to each HA alanine mutant compared to binding to the wild-type HA: red 25% binding; yellow 50–75% binding, blue 100% binding. Sequence numbering is based on H1N1 A/Puerto Rico/8/1934 coding region (GenBank accession number ABO21709). Subtypes that can be neutralized by PN-SIA49 are indicated with a green ‘+’ on the left, while the ones that can not be neutralized are indicate with a red ‘−’. ^a Recombinant HA from H1N1 A/South Carolina/1/1918 pandemic strain was previously shown to be bound by PN-SIA49 , . ^b H1N1 A/New Caledonia/20/1999 was previously shown to be neutralized by PN-SIA49 as Fab fragment , .

Figure 4. Amino acidic residues involved in the binding of PN-SIA49 to A/Puerto Rico/8/1934 (H1N1) hemagglutinin.

The HA of A/Puerto Rico/8/1934 is represented as trimer with the HA1 domains colored white and the HA2 domains colored cyan. Mutations that affect the binding of PN-SIA49 are highlighted in red.