Abstract
Cross-subtype neutralizing single domain antibodies against influenza present new opportunities for immunoprophylaxis and pandemic preparedness. Their simple modular structure and single open reading frame format are highly amenable to gene therapy-mediated delivery. We have previously described R1a-B6, an alpaca-derived single domain antibody (nanobody), that is capable of potent cross-subtype neutralization in vitro of H1N1, H5N1, H2N2, and H9N2 influenza viruses, through binding to a highly conserved epitope in the influenza hemagglutinin stem region. To evaluate the potential of R1a-B6 for immunoprophylaxis, we have reformatted it as an Fc fusion for adeno-associated viral (AAV) vector delivery. Our findings demonstrate that a single intramuscular injection in mice of AAV encoding R1a-B6 fused to Fc fragments of different isotypes equipped either, with or without antibody dependent cellular cytotoxicity (ADCC) activity, was able to drive sustained high-level expression (0.5–1.1 mg/mL) in sera with no evidence of reduction for up to 6 months. R1a-B6-Fc fusions of both isotypes gave complete protection against lethal challenge with both pandemic A/California/07/2009 (H1N1)pdm09 and avian influenza A/Vietnam/1194/2004 (H5N1). This data suggests that R1a-B6 is capable of cross-subtype protection and ADCC was not essential for R1a-B6 efficacy. Our findings demonstrate AAV delivery of cross-subtype neutralizing nanobodies may be an effective strategy to prevent influenza infection and provide long-term protection independent of a host induced immune response.
Highlights
Influenza virus continues to be a major public health concern, causing both annual epidemics and occasional pandemics [1]
We have previously described R1a-B6 as a potent alpaca derived nanobody capable of cross subtype neutralization of pandemic A(H1N1)2009, highly pathogenic avian influenza H5N1, H2N2 and H9N2 [28, 40]
Purified R1a-B6Fc fusion proteins were shown to bind to a broad panel of whole influenza virus reference reagents (Figure 1E and Supplementary Figure 1) and neutralize key Group I Influenza A subtypes, A(H1N1), A(H2N2), A(H5N1), and A(H9N2) (Supplementary Table 1)
Summary
Influenza virus continues to be a major public health concern, causing both annual epidemics and occasional pandemics [1]. Vaccines remain the main method of infection control, their timely implementation and poor immunogenicity in certain vulnerable patient groups remain a considerable problem [4]. Antiviral drugs such as Oseltamivir are available to control the spread of the virus their effectiveness is limited in treating patients with influenza [5, 6]. A much more promising strategy is to use recombinant monoclonal antibodies (mAbs) against influenza and several are currently in clinical development [9,10,11,12,13] These rare mAbs bind to functionally conserved epitopes such as those in the hemagglutinin (HA) stem, thereby providing strain independent protection. A more practical and cost-effective strategy would be to use antibody gene therapy which would provide long term sustainable protection through antibody production within the patient
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