Llama Nanoantibodies with Therapeutic Potential against Human Norovirus Diarrhea

Abstract

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

“…For example, in the case of HIV, with the aid of an extra long CDR3 loop, the neutralizing Nanobody D7 effectively competed for the CD4 binding site on gp120 protein [42]. Previously described Nanobodies and mAbs with therapeutic potential against human norovirus were also proposed to interfere with the HBGA binding site [20,[22][23][24][25][26][27]. MAb termed NV8812 bound to a conformational epitope on the GI.1 P domain and blocked the binding of norovirus VLPs to human and animal cell lines [24].…”

“…A recent study suggested that antibodies targeting the HBGA pocket could inhibit norovirus replication by steric interference with the GI.1 HBGA pocket [22]. A number of other studies have identified norovirus-specific monoclonal antibodies (mAbs) and single chain variable domains (VHH or Nanobodies) that could block norovirus VLP binding to HBGAs [20,[23][24][25][26][27]. However, most of these antibodies and Nanobodies are genotype specific, which limits their therapeutic potential [28].…”

Norovirus infection in primary immune deficiency

“…IFN‐λ is an attractive candidate for treatment of human norovirus infection because it has already been used as therapy for chronic HCV infection in Phase II clinical trials . An alternative approach for norovirus drug design is to target the binding of norovirus particles to HBGA carbohydrates, either using small compounds, monoclonal antibodies, or single chain antibody fragments . Therapies based on these approaches remain in the early stages of development.…”

Antiviral targets of human noroviruses

“…Human monoclonal antibodies that block HBGA binding have been isolated and produced from the peripheral blood mononuclear cells of blood donors, and the few that have been characterized to date appear to be genotype-specific [51]. In addition, nanobodies that block HBGA binding in GI.1 and GII.4 VLPs, have been identified and characterized [52*]. Currently the mechanism of how these antibodies block HBGA binding is unclear.…”