Her Majesty the Queen in Right of Canada, as Represented by the Minister of National Defence

Her Majesty the Queen in Right of Canada, as Represented by the Minister of National Defence of Her Majesty's Canadian Government

The Minister of Defence, Goverment of Canada

The Minister of National Defence, Government of Canada

Leslie P Nagata

Azhar Alvi

R Elaine Fulton

Weigang Hu

Jonathon P Wong

Wei-Gang Hu

Jonathan P H Wong

Josh Qiaohua Wu

A CDR grafted humanized recombinant antibody against infection from Venezuelan equine encephalitis virus (VEEV) comprises a human Ig framework having CDRs from murine mAb 1A4A1 VH and VL. DNA sequences, expression vectors incorporating such sequences and transformed host cells are also provided. Also provided are pharmaceutical compositions and methods of prophylaxis and treatment against VEEV infection using the humanized recombinant antibodies of the invention.

Humanized anti-Venezuelan equine encephalitis virus recombinant antibodies

This invention addresses how to rapidly prevent alphavirus-induced encephalitides before and after exposure to alphaviruses. The invention discloses a single dose administration of two types of recombinant viral vectors: one expressing interferon and another expressing the structural proteins of alphaviruses or a single dose administration of the recombinant viral vector co-expressing both interferon and the structural proteins of alphaviruses. This invention can be used to prevent humans from alphavirus-induced encephalitides in the event of a bioterrorism attack or biowarfare in which alphaviruses such as Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses are deliberately released to humans, a natural outbreak of alphaviruses, and an accidental exposure to alphaviruses in laboratory.

Inoculation of recombinant viral vectors for rapid pre-exposure prevention and post-exposure protection against alpha-virus-induced encephalitides

Construction of a recombinant gene fusion encoding a human IgG1 heavy chain constant region and a single-chain variable fragment antibody of 1A4A1 monoclonal antibody is disclosed. The recombinant antibody of the present invention confers human immune effector functions on murine antibodies. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody retains high antigen-binding affinity to VEE and possesses some human IgG crystallizable fragment domain functions. On non-reducing gel electrophoresis analysis, disulfide bond formation was found in the hinge region of the recombinant antibody. The present invention shows that the recombinant antibody is in a native, functionally active form and it provides the basis to characterize the recombinant antibody for efficacy in vivo.

Fusion protein of human IgG1 heavy chain constant region and scFv antibody against equine encephalitis virus

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being cloned into a prokaryotic T7 RNA polymerase-regulated expression vector was disclosed. A streptavidin-binding peptide (SBP) sequence fused to a 6His tag is then attached downstream to the scFv gene. The recombinant fusion protein is expressed in bacteria and then purified by immobilized metal affinity chromatography. ELISA and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody, but also possessed streptavidin-binding activity. This discovery obviates the need for chemical biotinylation of antibodies and the risk associated with antibody denaturation and provides a stable and reproducible reagent for rapid and efficient immunoassay of VEE.

Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus

A genetically biotinylated single chain fragment variable (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being applied in a system consisting of an immunofiltration-enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE is disclosed. The IFA entails formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capturing the sandwich by filtration on biotinylated membrane, and detecting the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies is investigated and optimized. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbant assay utilizing polystyrene plates and a chromogenic substrate, however, less time and effort were required for performance of the IFA/LAPS assay.

Genetically biotinylated recombinant antibody in immunofiltration assay by light addressable potentiometric sensor for identification of Venezuelan equine encephalitis virus

Construction and characterization of mouse monoclonal antibodies against western equine encephalitis virus (WEE) for potential use in detection, diagnosis, and immunotherapy are disclosed. Antibodies were prepared from hybridoma cells and further characterized by ELISAs, Western blotting, isotyping, and immunoprecipitation. The antibodies were also tested for cross-reactivity to other alphaviruses, such as Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), and eastern equine encephalitis (EEE). All antibodies bound to WEE antigen in ELISAs, whereas only a subgroup of antibodies was found to be active in Western blotting and immunoprecipitations. A subset of antibodies was found to cross-react with other alphaviruses, such as SIN, VEE, and EEE.

Construction and characterization of monoclonal antibodies against western equine encephalitis virus

This invention relates to the development of a mammalian expression vector, under which expression of the structural genes of western equine encephalitis virus have been placed under the control of an eucaryotic promoter. When the recombinant vector is administered to mammalian cell culture or using a cell-free transcription/translation system, in vitro, authentic structural proteins of western equine encephalitis virus are produced as verified by reactivity with monoclonal antibodies developed to western equine encephalitis virus. When the recombinant DNA molecule is administered in vivo, a protective immune response is induced, thereby enhancing protection of the individual against subsequent infection by western equine encephalitis virus. In a similar manner, DNA vaccines to related alphaviruses (Venezuelan and eastern equine encephalitis viruses) could also be developed.

DNA-based vaccine against the encephalitis alphaviruses

A single chain variable fragment (ScFv) antibody from a monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, is generated by cloning linked variable regions of the heavy (V ) and the light (V ) chain antibody genes. Mab clone 1A A in strain TG-1 was successfully cloned as ScFv. Results were reproduced in strain HB2151, expressing the same clone, A116, though displaying weak binding specificity to VEE due to a frame shift in the N-terminal region of the V domain, upstream to the complementarity-determining region 1. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5 prime region of the V gene of A116, corresponding to framework-1 region, producing mutant MA116, correcting a localized frame-shift in framework-1 region to consensus framework-1 amino acid sequence. A MA116 clone, MA116-15, shows comparable reactivity to the parental Mab in recognizing VEE antigen.

Cloning, expression, sequencing, and functional enhancement of monoclonal ScFv antibody against Venezuelan equine encephalitis virus (VEE)

Strain of the western equine encephalitis virus

The present invention relates to novel ribonucleotide oligonucleotides (RNOs) that are specifically designed to inhibit viral replication. The RNOs are capable of binding to both the negative and positive strands of influenza RNA segments, thereby inhibiting the virus' ability to produce various viral components, thus inhibiting viral propagation, and effectively killing the virus at the intracellular sites of infection in the respiratory tract. The RNOs provided may act independently, or in combination to optimize their antiviral activity. In addition, the RNOs provided may be formulated in liposomes, which facilitate their therapeutic delivery to intracellular sites of infection, and additionally increase antiviral efficacies.

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Medicine Hat, Canada

Leslie P Nagata