Creating Innovative Camelid Nanobody Approaches for Treatment of Severe Dengue

Dengue fever (DF) is a major threat to public health throughout the tropics. It is the most widespread vector-borne disease after malaria. Dengue is caused by four dengue virus serotypes (DENV-1, -2, -3, -4). The immunopathogenesis of dengue disease remains elusive, with various mechanisms postulated including memory immune cell responses, antibody enhancement, host genetic factors, and the ability to damage endothelial cell barriers in addition to inherent factors of the virus. Dengue can progress rapidly from benign febrile illness to severe dengue (HF/DSS) and death. Early diagnosis is important to identify those patients who may develop or already have Severe disease to target supportive therapies. However, there are no U.S. Food and Drug Administration-approved anti-DENV countermeasures. We hypothesize that anti-DENV Nanobodies (Nbs) to envelope (Env) and non-structural-1 (NS-1) proteins will neutralize and protect against DENV infection and microvascular endothelial cell (MEC) barrier dysfunction. Small in size, Nbs allow for binding into the groves and sites, which are generally not accessible to antibodies, thereby increasing the epitope repertoire on an antigen available for binding with the Nbs. This property of Nbs can therefore enhance the specificity of Nb-based therapeutics. We have extensive experience in camelid Nb generation. Functional camelid IgG2 and IgG3 antibodies (Abs) lack light chains. The Nb is the variable heavy chain (VHH) fragment of the camelid Ab. Nbs offer little immunogenicity, high solubility, stability at low/high pH, activity against tumor cells, and recognize hidden antigenic sites. Camelid Nbs targeting can act as prophylactic and therapeutic drugs against viral infections. Targeting the most acute and often fatal consequences of DENV infection, we predict that Nbs targeting the dengue Env protein will have an adjunct role in rapidly neutralizing free virus, while Nbs targeting NS-1, arguably the major trigger for fatal Severe dengue associated microvascular dysfunction, will rapidly remove this protein from the circulation. Nbs can bind to hidden antigenic sites that are not accessible to conventional antibodies and provide a wider repertoire of neutralizing Nbs including against epitopes that are still not known. Thus, our main approach will be to create Nbs to intact lethally irradiated DENV in the presence of a radio-protective Mn2+-Peptide complex from Deinococcus (MDP-iDENV) to preserve virion epitope (including M/capsid protein) immunogenicity. We hypothesize that this non-biased approach will provide therapy/diagnostic targeting different sites, known or not, on mature virions. A combination therapy that include Nbs to viral proteins required for replication will provide a better therapeutic response to the virus than targeting a single moiety or protein on the virus. Specific Aims: Aim 1: To generate irradiation inactivated DENV with preserved antigen structure (MDP-iDENV) and create nanobody libraries from alpaca co-immunized with serotype matched MDP-iDENV and recombinant DENV non-structural protein-1 (NS-1). Aim 2: Non-biased in vitro "functional screens" to assess therapeutic efficacy to block cell infection, and control hypovolemic shock and thrombo-inflammation. This will be done by (i) electric cell-substrate impedance sensing (ECIS) and to measure their ability to reverse MEC barrier dysfunction, and detection of potential Nb cytotoxicity for MEC barriers; (ii) to block cell infection by plaque reduction neutralization test (PRNT); and (iii) to minimized infection induced MEC inflammatory responses. Aim 3: Preclinical Nb testing in human-immune-system humanized DRAGA murine models challenged with DENV. Humanized DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RgammacKO.NOD) HLA-expressing mice reconstitute a functional human-immune-system found suitable as models for human malaria and COVID-19. Study Highlights: • Creation of mitigation strategies to protect military and civilian personnel against the severest clinical manifestations of dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). • Generation of dengue virus (DENV) serotypes lethally gamma-irradiated in the presence of a radio-protective Mn2+-Peptide complex from Deinococcus (MDP-iDENV) to preserve epitope immunogenicity and function. • Generation of nanobodies (Nbs) from alpaca co-immunized with MDP-iDENV and DENV serotype-matched recombinant non-structural protein 1 (NS1). • Non-biased in vitro "functional screens" for Nb therapeutic efficacy: (i) by neutralization assay; (ii) by electric cell-substrate impedance sensing (ECIS) for microvascular barrier function and Nb cytotoxicity; and (iii) assays for inflammatory potential. • Preclinical testing in human-immune-system humanized DRAGA murine models challenged with DENV serotypes. Less