WO2016149089A1 - Vaccin composé de sous-unités pour la prévention d'une maladie causée par l'astrovirus - Google Patents

Vaccin composé de sous-unités pour la prévention d'une maladie causée par l'astrovirus Download PDF

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WO2016149089A1
WO2016149089A1 PCT/US2016/021987 US2016021987W WO2016149089A1 WO 2016149089 A1 WO2016149089 A1 WO 2016149089A1 US 2016021987 W US2016021987 W US 2016021987W WO 2016149089 A1 WO2016149089 A1 WO 2016149089A1
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hastv
astrovirus
spike
capsid
vaccine
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Rebecca DUBOIS
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/12011—Astroviridae
    • C12N2770/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/12011—Astroviridae
    • C12N2770/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Described herein are methods and compositions related to vaccination and immune development strategies for prevention of astrovirus disease causing intestinal disease and conditions such as diarrhea.
  • HstV Human astrovirus
  • a protective HAstV vaccine would significantly benefit human health by preventing millions of cases of childhood gastroenteritis worldwide and would also reduce economic burden associated with medical care and absence from work by parents caring for HAstV-infected children.
  • Described herein is the discovery that a single domain in the HAstV capsid protein plays a key role in binding to a potent neutralizing antibody. Furthermore, structural and mechanistic studies on both the HAstV capsid domain and the neutralizing antibody reveal the atomic interactions and mechanism of action of the neutralizing antibody targeting the HAstV capsid protein, thereby providing a means to develop a HAstV capsid subunit vaccine by uncovering a point of vulnerability on the HAstV virus capsid surface.
  • a vaccine for protecting a mammal against a disease condition resulting from an astrovirus infection including a subunit of an astrovirus and an adjuvant.
  • the subunit of an astrovirus includes a capsid protein.
  • the subunit of an astrovirus includes a capsid protein spike.
  • the capsid protein spike includes a receptor binding domain.
  • the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the disease condition includes diarrhea or gastroenteritis.
  • the vaccine includes subunit of and an adjuvant.
  • the subunit of an astrovirus includes a capsid protein.
  • the subunit of an astrovirus includes a capsid protein spike.
  • the capsid protein spike includes a receptor binding domain.
  • the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the disease condition includes diarrhea or gastroenteritis.
  • the one or more astrovirus proteins comprise a capsid protein.
  • the capsid protein includes a capsid protein spike.
  • the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the cells are eukaryotic.
  • the cells are prokaryotic.
  • the astrovirus protein includes a histidine tag.
  • the histidine tag includes a C-terminal or N-terminal tag.
  • the one or more astrovirus proteins comprise a capsid protein.
  • the capsid protein includes a capsid protein spike.
  • the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the cells are eukaryotic.
  • the cells are prokaryotic.
  • the astrovirus protein includes a histidine tag.
  • the histidine tag includes a C-terminal or N-terminal tag.
  • inducing an immune response against astrovirus including administering one or more astrovirus proteins to a mammal.
  • the one or more astrovirus proteins comprise a capsid protein spike derived from human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • inducing an immune response includes production of one or more antibodies specific to one or more astroviruses.
  • the one or more antibodies comprise a monoclonal antibody.
  • the one or more antibodies comprise a polyclonal antibody.
  • FIG. Astroviruses infection of mammals and birds.
  • FIG. 2A Cryo-electron microscopy image of mature astrovirus virion composed of the mature capsid protein. Arrow points to one of the 30 dimeric spikes on the surface of the virus.
  • FIG. 2B High-resolution crystal structures of HAstV strain 8 and turkey astrovirus strain 2 capsid spikes
  • FIG. 2C Proteolytic processing and maturation of the astrovirus capsid protein and recombinant HAstV capsid spike construct.
  • FIG. 3A Recombinant HAstV capsid domains produced for these studies
  • FIG. 3B SDS-PAGE and Western Blot showing reactivity to anti-HAstV-1 rabbit serum polyclonal antibodies (generated by HAstV-1 virus immunization). Molecular weight markers (MW, in kD), Shell (lane 1), Spike (lane 2), Acidic (lane 3).
  • Fig. 3C ELISA showing binding to both HAstV capsid Shell and Spike domains. Loading controls are tested with an anti-His-tag antibody. Negative controls have no primary antibody.
  • FIG. 4A Protein G purification of MAb PL-2.
  • FIG. 4B HAstV-2 capsid spike, but not HAstV-1 capsid spike (Fig. 4C) or HAstV-1 shell (Fig. 4D) domains, binds MAb PL-2 by ELISA.
  • FIG. 5A High-resolution crystal structures of recombinant HAstV-1 and HAstV-2 capsid spike.
  • FIG. 5B HAstV-2.
  • HAstV capsid spike binds to Caco-2 cells in a specific manner.
  • Caco-2 cells were treated with 5 to 10 ⁇ of EGFP and EGFP-Spike for 1 hour or 24 hours, followed by extensive washes. Live cells were visualized by confocal microscopy. Plasma membranes were labeled with Alexa Fluor 594 - conjugate wheat germ agglutinin (red) and the nuclei were labeled with Hoechst 33342 (blue).
  • Fig. 6A EGFP
  • Fig. 6B Plasma membrane and nuclear
  • Fig. 6C Merger image of EGFP, plasma membrane, and nuclear. Negative control EGFP samples appeared very similar at the 1 hour time point (not shown) to the 24 hour time point.
  • FIG. 7 Spike sequence alignment and conserved residues mapped onto the 0.9 A preliminary structure of HAstV- 1 spike.
  • Fig. 7A HAstV- 1-8 capsid spike sequence alignment produced by ESPript.
  • B C. Preliminary HAstV-1 capsid spike structure shown from top (Fig. 7B) and side (Fig. 7C) Half of the dimer is grey, and the other half is green. conserveed amino acids are colored red, and homologous residues are pink. Two patches of conserved residues are circled in blue or red.
  • HAstV receptor-binding spike residues may be inaccessible on the immature HAstV.
  • Fig. 8 A Cryo-EM model of immature HAstV and inaccessibility of receptor-binding site candidates due to steric hindrance with neighboring spikes.
  • Fig. 8B Cryo-EM model of mature HAstV and accessibility of receptor-binding site candidates.
  • FIG. 9 Glycan microarray analyses with recombinant HAstV- 1 capsid spike. HAstV-1 spike at pH 7 (left) and at pH 3 (right). The data are linear to a maximum RFU of -50,000. In all cases, no significant binding was observed. Glycans 81 (Fucal-4GlcNAcb- Sp8) and 451 (Galal-3(Fucal-2)Galbl-4GlcNAcbl-6(Galal-3(Fucal-2)Galbl4GlcNAcbl- 3)GalNAc-Spl4) were not considered positive because they are known to have non-specific interactions with many proteins that are not glycan-binding proteins.
  • Glycans 224 (Neu5Aca2-3Galbl-3GalNAca-Sp8) and 265 (Neu5Aca2-3Galbl-4Glcb-Sp8) were also not considered significant due to the low signal and the lack of binding by related glycans on the microarray.
  • FIG. 10 Far Western Blot reveals EGPP-HAstV-1 capsid spike binding to discrete Caco-2 cell proteins.
  • Fig. 10A Recombinant EGFP (lane 1) and EGFP-Spike (lane 2) used in fluorescence microscopy assays and Far Western Blot assays. Proteins were purified by affinity and size-exclusion column chromatography.
  • Fig. 10B Far Western Blot to detect EGFP-Spike and EGFP binding to a specific Caco-2 cell proteins. One, 5, or 10 ⁇ g of Caco-2 whole cell lysate was loaded onto each lane as indicated above.
  • BSA-blocked nitrocellulose membranes were incubated with 5 ⁇ of EGFP-Spike or 5 ⁇ EGFP overnight at 4°C. Proteins were detected by HRP-conjugated Anti-His-tag antibody. The red bracket indicates proteins specifically bound by EGFP-Spike but not EGFP.
  • FIG. 11 Fab PL-2 and HAstV-2 capsid spike bind in a 2:2 stoichiometric complex.
  • FIG. 11 A Size-exclusion chromatography traces with MW standards (top) or mixtures of Fab PL-2 and spike (bottom).
  • FIG. 11B Samples visualized by reducing SDS-PAGE. MW: MW Markers. Lane 2: Spike. Lane 3 : Fab PL-2. Lane 4: Fab/spike complex.
  • FIG. 11C Model of Fab/spike binding studies.
  • FIG. 11D Experimental design utilizing excess spike or antibody to confirm stoichiometry and binding effects.
  • FIG. 12 Fab PL-2 and HAstV-2 capsid spike crystals and preliminary high- resolution structures.
  • Fig. 12A HAstV-2 capsid spike and structure.
  • Fig. 12B Fab PL-2 spike and structure.
  • FIG. 13 Production of recombinant scFv PL-2 in Schneider 2 insect cells. Elutions of scFv PL- 2 from Strep-tactin affinity chromatography column are shown highlighted in yellow stars.
  • FIG. 14A Schematic of HAstV-1 capsid protein domain structure and proteolytic processing/maturation events.
  • Caspase and trypsin cleavage sites are indicated with white and orange arrows, respectively.
  • Fig. 14B, Fig. 14C Structures of HAstV-1 capsid shell (Fig. 14B) and spike (Fig. 14C) domains.
  • Fig. 14D, Fig. 14E Models of immature (Fig. 14D) and mature (Fig. 14E) HAstV-
  • HAstV capsid spike is the main antigenic domain.
  • Fig. 15A SDS-PAGE and Western Blot showing reactivity to anti-HAstV-1 rabbit serum polyclonal antibodies.
  • Molecular weight markers (MW, in kD), Shell (lane 1), Spike (lane 2). Fig. 15B, 15C.
  • ELISA showing binding of a-HAstV-1 polyclonal sera (diluted in series 1 :4) to recombinant HAstV-1 capsid shell (Fig. 15B) and spike (Fig. 15C). Little or no cross-reactivity is observed with sequence divergent turkey AstV (TAstV-2) capsid domains. Blank controls have no primary polyclonal antibody.
  • FIG. 16 Mice immunized with recombinant HAstV-1 capsid spike develop a HAstV-1 -neutralizing antibody response.
  • Caco-2 cell monolayers were infected with HAstV- 1 that had been previously incubated with serial dilutions of sera raised in mice to the HAstV capsid spike domain (Spikel) or to the complete purified HAstV-1 virus.
  • the cells were fixed, permeabilized, and incubated with rabbit anti-HAstV serum, followed by anti-rabbit IgG antibodies coupled to Alexa 488. Infected cells were analyzed by fluorescence microscopy. The dilution of the sera is indicated. The control virus was incubated with preimmune mouse serum.
  • FIG. 17 HAstV capsid spike binds to Caco-2 cells and binding is blocked by scFv PL-2.
  • Fig. 17A Coomassie-stained SDS-PAGE. MW, Molecular weight markers (MW). 1, EGFP. 2, EGFP-Spike2. 3, EGFP with 4 molar excess scFv PL-2. 4, EGFP-Spike2 with 4 molar excess scFv PL-2. 5, EGFP- Spike2-A Site 1.
  • Fig. 17B, Fig. 17C FACS assay data showing fluorescence of Caco-2 cells incubated 18 h with recombinant proteins.
  • Fig. 17D Live Caco-2 cells visualized by fluorescence microscopy.
  • Fig. 17E Fixed Caco-2 cells visualized by confocal microscopy 18 h after addition of EGFP-Spike. Plasma membranes were labeled with AlexaFluor594 - conjugate wheat germ agglutinin (red) and nuclei were labeled with Hoechst stain (blue).
  • human astrovirus is a leading cause of viral gastroenteritis in children, and is also attributed to chronic gastroenteritis in hospitalized or immune- compromised children as well as the elderly.
  • Astroviruses can also cause infections and disease in other mammalian and avian animals.
  • astrovirus is associated with growth defects and mortality in poultry as well as encephalitis in cows. No licensed vaccines or antiviral therapies exist for HAstV infection.
  • the capsid protein undergoes intracellular and extracellular protease processing required for mature virus formation and infectivity.
  • HAstV is classified into serotypes, where HAstV-1 is the predominant strain worldwide.
  • the mature astrovirus capsid is the target of previously described neutralizing antibodies.
  • Two studies isolated monoclonal antibodies (MAbs) against HAstV that neutralize astrovirus infection in cell culture, including .
  • HAstV is an understudied pathogen.
  • HAstV growth in cell culture requires specific human or primate cell lines. Human colon carcinoma Caco-2 cells being the most widely used, but HAstV does not grow in commonly-used biopharmaceutical cell lines for GMP production such as CHO, MDCK, BHK-21, and grows poorly in HEK-293 cells.
  • HAstV virions are highly stable and resistant to chemical and UV inactivation, suggesting that virus inactivation may be challenging.
  • highly-UV-inactivated HAstV is no longer able to replicate and cause an infection, it still has properties that cause it to open epithelial cell tight junctions, suggesting that an inactivated HAstV may still cause disease or have side effects.
  • production of a live attenuated HAstV vaccine is not possible at this time because an animal model for HAstV (human) does not exist, thus no method exists to test for an attenuated HAstV vaccine's ability to provide protection without causing disease.
  • Astrovirus capsid protein is a multi-domain protein that spontaneously assembles into immature particles, which then undergo a series of intracellular and extracellular proteolytic cleavages that are required for mature virus formation, virus release, and virus infectivity.
  • a major challenge in identifying the HAstV capsid receptor-binding site is simply production of functional recombinant HAstV capsid protein, a large multi- domain protein that assembles into heterogeneous virus-like particles and undergoes both intracellular and extracellular proteolytic cleavages during HAstV maturation.
  • the HAstV capsid spike composes a RBD.
  • the location of the spike as the outermost domain on the surface makes it a logical option, and the spikes of many other non-enveloped viruses are receptor-binding domains.
  • the spikes of many other non-enveloped viruses are receptor-binding domains.
  • high divergence in capsid spike sequences between astroviruses that infect different species suggest that there is a species-specific receptor that only binds to the spike of the astrovirus that infects that species.
  • immunoassays by neutralizing MAbs that block HAstV attachment to cells were found to immunoprecipitate 25-29kD capsid fragments, which are now known compose the spike domain fragments.
  • capsid protein assembles into virus-like particles, undergoes intracellular caspase cleavage and extracellular trypsin cleavage, but can only be partially purified in limited ⁇ g) quantities.
  • vaccine production strategy uses standard bacterial cell lines that are already used in therapeutic protein production, thus low side effects are expected.
  • HAstV capsid subunit antigen can be injected into a patient, whose adaptive immune system would elicit a protective antibody response that results in life-long protection against HAstV infection.
  • Critical to this process is molecular understanding of the human astrovirus (HAstV) capsid' s roles in virus attachment to human cells, antibody neutralization, and immunogenicity.
  • the HAstV capsid spike domain binds to a specific human cell surface receptor and also elicits and binds HAstV-neutralizing antibodies, this feature of HAstV represents the "Achilles' heel" of HAstV. Further exploiting such key vulnerabilities on the HAstV capsid surface will provide a foundation for the development of a fully protective HAstV vaccine. As described, designing an effective HAstV subunit vaccine requires characterization of the location of functional sites and neutralization epitopes on the HAstV capsid surface.
  • Gap in knowledge about these sites can be attributed to the challenges in studying the HAstV capsid protein, a large multi-domain protein that assembles into virus-like particles and undergoes both intracellular and extracellular proteolytic cleavages during HAstV maturation.
  • the Inventors have taken an innovative approach by producing HAstV capsid protein as individual recombinant capsid structural domains.
  • the Inventors have developed novel receptor-binding assays and have also acquired the unique HAstV-neutralizing monoclonal antibody PL-2.
  • the Inventors provide structural, biochemical, cellular, and immunological evidence that the HAstV capsid spike domain composes a receptor-binding domain and contains a neutralizing epitope. Understanding atomic resolution and these binding interactions identify the key vulnerabilities of HAstV that can be exploited for the development of HAstV vaccine immunogens and antiviral therapeutics.
  • a blueprint for the design and production of immunogens that elicit broadly neutralizing antibodies help identify those features that provide protection in animal models of HAstV infection.
  • a protective HAstV vaccine would significantly benefit human health by preventing millions of cases of childhood gastroenteritis worldwide.
  • a protective HAstV vaccine would also reduce economic burden associated with medical care and absence from work by parents caring for HAstV-infected children.
  • the Inventors' atomic resolution insight into key HAstV vulnerabilities will also help advance the development of antiviral therapeutics for immune- compromised patients with severe or persistent HAstV infection.
  • the Inventors will enable virologists to build upon the Inventors' molecular insights and study their broader implications in astrovirus pathogenesis, including testing the role of the cell surface receptor(s) in HAstV infection, testing the role of the spike receptor-binding and/or neutralization site in HAstV infection, and testing the correlation between serum antibodies targeting HAstV capsid spike and protection from HAstV disease.
  • structurally characterizing the molecular interactions between a protective neutralizing antibody and the virus surface capsid protein one can validate and expand structural findings with established and novel biochemical and cell-based assays. These studies help reveal sites of vulnerability on the astrovirus capsid protein that can be exploited for design of an effective astrovirus vaccine.
  • visualizing in molecular detail how viruses enter and replicate in human cells one can use this information to develop new vaccines and antiviral therapeutics.
  • structure-based vaccine design uses techniques such as X-ray crystallography and electron microscopy, to visualize the molecular structures of virus surface proteins alone, bound to human cell surface receptors, and bound to neutralizing antibodies. Analyses of these molecular structures will allows one to establish structurally informed biochemical and cell-based experiments to elucidate the key molecular interactions between virus and host. Critical to the descried processes is relying on information derived from these studies to engineer virus surface proteins as effective vaccine antigens that elicit virus-neutralizing antibodies. Extending those results, one can also deploy structure-based drug discovery focuses on how viruses replicate in human cells and how small molecule therapeutics can block this activity.
  • virus RNA polymerase proteins required for both the replication of virus genome and transcription of virus mRNA.
  • Virus RNA polymerases are essential for virus survival and usually have high sequence similarity between different strains in a virus family, making these proteins ideal targets for antiviral drug development. Protein engineering, X-ray crystallography, high-throughput biochemical screening, and virology allow identification of high-affinity and high-specificity therapeutics that block virus replication involving these molecules.
  • biochemical assays can confirm and extend understanding of the molecular mechanisms of astrovirus neutralization.
  • the elucidated molecular structure can guide the design of an astrovirus capsid fragment as a novel vaccine antigen to elicit high levels of broadly neutralizing antibodies that protect against astrovirus infection.
  • HAstV-1 capsid spike crystallized, and solved the structure of the capsid spike.
  • ELISA enzyme-linked immunosorbent assay
  • MAb PL-2 Fab fragments and solve structure.
  • the pursuit of crystallographic studies requires that macromolecular samples be highly pure, highly concentrated, structurally homogenous, and ideally conformationally rigid.
  • elucidating the structure of the full-length MAb PL-2 may not be dispositive as MAbs in general are quite flexible at the hinge region.
  • HAstV-2 spike dimer binds 1 or 2 Fab PL-2 fragments. It is conceivable that the spike dimer (2 molecules) would bind two Fab PL-2 fragments. However, it is equally conceivable that the spike dimer may only have one epitope that spans the dimerization interface, and thus would only bind one Fab fragment. To ascertain binding structure, one can mix Fab PL-2 and HAstV-2 spike, in ratios with either excess Fab or excess spike. Samples are analyzed by size-exclusion chromatography and polyacrylamide gel analyses. Such binding interactions will guide approaches for designing immunogens for vaccine development.
  • BIAcore surface plasmon resonance to measure rates of binding and disassociation. Overall, these assays will confirm sites of vulnerability and neutralization. Thereafter, one can utilize a cell-based receptor-binding assay with labeled GFP-HAstV spike.
  • An assay to test HAstV spike receptor-binding activity using a fluoresecent reporter allows one to test binding of spike wild-type and musants and probe the identity of host cell receptor. Although the HAstV host cell receptor is unknown, HAstV attachment to human cells (receptor-binding) is reported to be blocked by neutralizing MAbs.
  • the assay described herein involves incubation of purified fluorescently labeled spike (GFP-HAstV spike) with Caco-2 colon cells, which are susceptible to HAstV infection, and examination of cells by fluorescence microscopy.
  • GFP-HAstV spike purified fluorescently labeled spike
  • Caco-2 colon cells which are susceptible to HAstV infection
  • MAb PL-2 binds to MAb PL-2
  • a recent study identified binding between HAstV capsid and fibronectin 1 receptor, and the described assay can also explore this reported binding further. Overall, these studies will reveal the mechanism of MAb PL-2 neutralization and sites of vulnerability on HAstV spike.
  • rabbits can be immunized with purified HAstV capsid spike antigen and serum collected to test the antigenic properties and vaccine potential of HAstV capsid spike antigen.
  • purified, endotoxin-free, recombinant HAstV-1 spike, pre- and post-immune serum will be collected Serum is then tested for HAstV capsid spike-specific antibodies and neutralizing activity.
  • Serum antibody titer can be measured using (1) ELISA to measure serum antibody titer, (2) cell-based receptor-binding assay with GFP-HAstV spike to test for serum antibodies that block receptor-binding, (3) measurement of serum neutralizing antibody titer against HAstV-1 growing in Caco-2 cells. Serum neutralizing antibody titer against other strains (HAstV-2-8) can broadly establish neutralizing antibody titers against various virus strains.
  • the subunit of an astrovirus includes a capsid protein.
  • the subunit of an astrovirus includes a capsid protein spike.
  • the subunit of an astrovirus includes HAstV- 1 spike, for example amino acids 430 to 648 of the HAstV- 1 protein, or HAstV-2 spike, for example amino acids 430 to 645 of the HAstV-2 protein.
  • the capsid protein spike includes a receptor binding domain.
  • the astrovirus includes human astrovirus (HAstV)- 1, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc.
  • the disease condition includes diarrhea or gastroenteritis.
  • the vaccine includes subunit of and an adjuvant.
  • the subunit of an astrovirus includes a capsid protein.
  • the subunit of an astrovirus includes a capsid protein spike.
  • the subunit of an astrovirus includes HAstV-1 spike, for example amino acids 430 to 648 of the HAstV-1 protein, or HAstV-2 spike, for example amino acids 430 to 645 of the HAstV-2 protein.
  • the capsid protein spike includes a receptor binding domain.
  • the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc.
  • the disease condition includes diarrhea or gastroenteritis.
  • polyclonal antisera against HAstV-1, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8 is isolated from a mammal as produced by the method of administering a vaccine.
  • compositions including cells capable of producing one or more astrovirus proteins.
  • the one or more astrovirus proteins comprise a capsid protein.
  • the capsid protein includes a capsid protein spike.
  • the subunit of an astrovirus includes HAstV-1 spike, for example amino acids 430 to 648 of the HAstV-1 protein, or HAstV-2 spike, for example amino acids 430 to 645 of the HAstV-2 protein.
  • the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc.
  • the cells are eukaryotic.
  • the cells are prokaryotic.
  • the cells are mink or chicken cells.
  • the cells are insect cells.
  • the astrovirus protein includes a histidine tag.
  • the histidine tag includes a C-terminal or N-terminal tag.
  • the astrovirus protein includes a fluorescent label such as green fluorescent protein (GFP).
  • the method of producing one or more astrovirus proteins using includes a composition including cells capable of producing one or more astrovirus proteins.
  • the one or more astrovirus proteins comprise a capsid protein.
  • the capsid protein includes a capsid protein spike.
  • the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc.
  • the cells are eukaryotic.
  • the cells are prokaryotic.
  • the astrovirus protein includes a histidine tag.
  • the histidine tag includes a C-terminal or N-terminal tag.
  • the astrovirus protein includes a fluorescent label such as green fluorescent protein (GFP).
  • the one or more astrovirus proteins comprise a capsid protein spike derived from human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.
  • the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc.
  • inducing an immune response includes production of one or more antibodies specific to one or more astroviruses.
  • the one or more antibodies comprise a monoclonal antibody.
  • the one or more antibodies comprise a polyclonal antibody.
  • Astrovirus capsid protein is a multi-domain protein that intracellularly assembles into immature virus particles, which then undergo a series of intracellular and extracellular proteolytic cleavages that are required for mature virus formation, virus release, and virus infectivity (Fig. 2).
  • Recombinant astrovirus capsid spike domain is produced by cloning cDNA encoding the capsid spike domain into an expression plasmid for production in E. coli, similar to previously published studies.
  • the Inventors have establish a means for easy purification, however, other purification strategies, including N-terminal purification tags, are also feasible.
  • Affinity and size-exclusion chromatography purification steps yield highly pure protein (25kD), as assessed by SDS-PAGE (Fig. 3B, lane 2) which elutes as a ⁇ 50kD dimer on a size-exclusion chromatography column in comparison to standard proteins.
  • Purified, recombinant HAstV capsid spike is recognized by polyclonal antibody serum (generated against HAstV virions) by both Western Blot and ELISA (Fig. 3B, 3C).
  • MAbs neutralizing monoclonal antibodies
  • MAb PL-2 neutralizing monoclonal antibodies
  • the Inventors produced recombinant HAstV-2 spike and found that it bound strongly to pure MAb PL-2 by ELISA (Fig. 4B). Consistent with the reported specificity for serotype HAstV- 2, the Inventors found that MAb PL-2 did not bind HAstV- 1 spike or shell domains (Fig. 4C, 4D).
  • the HAstV capsid spike domain is the virus's receptor-binding (i.e. cell attachment) domain.
  • RBD receptor-binding domain
  • the location of the spike as the outermost domain on the surface makes it a logical option, and the spikes of many other non- enveloped viruses are receptor-binding domains.
  • high divergence in capsid spike sequences between astroviruses that infect different species suggest that there is a species-specific receptor that only binds to the spike of the astrovirus that infects that species.
  • immunoassays by neutralizing MAbs that block HAstV attachment to cells were found to immunoprecipitate 25-29kD capsid fragments, which are now known to compose the spike domain fragments.
  • the Inventors have developed a novel fluorescence microscopy assay to directly visualize HAstV capsid spike attachment and endocytosis into cells.
  • the Inventors produced a recombinant fusion protein composed of enhanced green fluorescent protein (EGFP) fused at the N-terminus of HAstV-1 capsid spike (EGFP-Spike).
  • EGFP-Spike enhanced green fluorescent protein
  • Inventors then incubated the purified recombinant proteins for short (1 hour) and long (24 hour) time points with Caco-2 human colon carcinoma cells (Caco-2 cells), the gold-standard cell line for HAstV propagation. Cells were washed thoroughly followed by live-cell visualization by confocal microscopy. The Inventors find that EGFP-Spike binds specifically to the surface of Caco-2 cells at the 1-hour time point, consistent with it being a HAstV receptor-binding domain (Fig. 6). Interestingly, EGFP-Spike localizes to specific regions of the Caco-2 cell surface in a punctate pattern, suggestive of a targeted binding event.
  • EGFP-Spike appears to be localized inside cells. This apparent localization, possibly in endosomes, is consistent with recent studies showing HAstV cell entry via endocytosis. Together, the Inventors' data suggest that the HAstV capsid spike is a receptor-binding domain, and neutralizing antibodies may block HAstV infection by blocking host cell attachment.
  • Recombinant HAstV capsid spike forms a well-folded, dimeric structure.
  • Recombinant HAstV capsid spike mimics the HAstV virus surface in that it is recognized by polyclonal antibodies raised against infectious HAstV virions.
  • the recombinant HAstV capsid spike is the target of a potent neutralizing monoclonal antibody.
  • recombinant HAstV capsid spike is a HAstV receptor-binding domain.
  • HAstV-neutralizing antibodies binding the HAstV capsid spike may function by blocking virus attachment to human cells
  • HAstV subunit vaccine composed of the spike domain will elicit anti -HAstV neutralizing antibodies.
  • recombinant HAstV- 1 capsid spike antigen will be an effective immunogen and elicit high levels of anti-HAstV neutralizing antibodies.
  • a particular focus will be for a vaccine for serotype HAstV- 1 as the predominant strain worldwide among eight canonical human astrovirus serotypes (HAstV-1-8).
  • the Inventors will use rabbit antibody production with standard immunization protocols to test the immunogenic properties of HAstV- 1 capsid spike.
  • Rabbits Three rabbits are immunized with purified, endotoxin-free, recombinant HAstV- 1 capsid spike, pre- and post-immune sera will be collected. Rabbits are ideal because (1) rabbits will provide sufficient volume of post-immune serum for further experiments, and (2) rabbits are being considered as an animal model for astrovirus-induced gastroenteritis. One then tests serum for antibodies that bind HAstV- 1 capsid spike by ELISA, and can also to test for neutralizing antibodies that block HAstV- 1 replication in Caco-2 cells.
  • subunit vaccine strategy could be used to develop an astrovirus vaccine against any strain of astrovirus, including those which cause disease in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc.
  • HAstV attaches to human cells via specific interactions between a conserved HAstV capsid spike receptor-binding site and a cell surface receptor.
  • the Inventors developed a novel fluorescence microscopy assay to show that the HAstV capsid spike binds to the surface of Caco-2 cells and becomes endocytosed.
  • the Inventors also solved the 0.9 A structure of the HAstV capsid spike, which forms a dimer, and have identified conserved amino acids that may compose a receptor-binding site.
  • HAstV capsid spike binds a proteinaceous cell surface receptor. Complete elucidation of these interactions involves: (1) Structurally and biochemically characterizing the HAstV capsid spike receptor-binding site. (2) Biochemically identifying HAstV capsid spike cell surface receptor candidate(s). (3) Biochemically and genetically validating the HAstV capsid spike cell surface receptor(s).
  • Certain HAstV-neutralizing antibodies function by blocking the receptor-binding site on the HAstV capsid spike and it is suggested that a recombinant HAstV capsid spike immunogen will elicit these antibodies.
  • the Inventors have discovered that the HAstV- neutralizing monoclonal antibody PL-2 binds strongly to the HAstV capsid spike.
  • the Inventors have also solved the 1.9 A structure of the PL-2 Fab fragment and have determined its de novo amino acid sequence.
  • This atomic resolution of binding interaction allows one to: (1) Structurally characterize the site of HAstV neutralization by monoclonal antibody PL-2. (2) Biochemically and mechanistically characterize the HAstV capsid spike site of neutralization. (3) Characterize immunogenicity of recombinant dimeric HAstV capsid spike immunogen.
  • HAstV capsid spike domain produced in E. coli retains functional and antigenic properties and can be produced in significant amounts (mgs) and to high purity for X-ray crystallographic, mechanistic, and vaccine studies.
  • HAstV receptor-binding assay using purified GFP-HAstV capsid spike allows one to easily generate receptor-binding-inactive spike mutants not limited by the necessity to amplify infectious HAstV.
  • Traditional methods to study HAstV attachment and entry involve incubation of infectious HAstV virions with human cells, followed by cell fixation, permeabilization, and immunoperoxidase visualization of virions.
  • a reverse genetics system for HAstV has been established and could be used to generate HAstV mutant virus, recovered virus titers are low and could not be amplified with receptor-binding-inactive mutations.
  • structure-based vaccine design allows for the design of improved immunogens that could not be obtained by traditional methods.
  • HAstV vaccine generation by traditional methods such as virus attenuation or inactivation is challenging due to the HAstV stability and resistance to chemical and UV treatment.
  • HAstV attaches to human cells via specific interactions between a conserved HAstV capsid spike receptor binding site and a cell surface receptor.
  • the Inventors' lab and others have previously determined the crystal structures of the human and avian astrovirus spikes (Fig. 2B).
  • Newly synthesized HAstV capsid proteins are multidomain proteins that spontaneously assemble into immature particles, which then undergo a series of intracellular and extracellular proteolytic cleavages that are required for virus release and infectivity (Fig. 2C).
  • the mature HAstV virion attaches to human cells via an unknown cell surface receptor and gains entry via clathrin-mediated endocytosis.
  • HAstV serotype 1 capsid spike domain as a recombinant protein in E. coli
  • the Inventors produced the HAstV serotype 1 (HAstV- 1) capsid spike domain as a recombinant protein in E. coli.
  • the Inventors chose to investigate serotype HAstV-1 because it is the predominant serotype worldwide among eight canonical serotypes (HAstV- 1-8).
  • the Inventors know that the recombinant HAstV- 1 spike is folded correctly because the Inventors have crystallized it and determined the 0.9 A structure of the HAstV-1 spike by X-ray crystallography (Fig. 2).
  • the dimeric spike is nearly identical in its structural fold (0.5 A RMSD) compared to the previously determined HAstV-8 spike structure.
  • HAstV-1-8 serotypes use the same cell surface receptor and have conserved amino acids that compose a receptor-binding site. The evidence from this comes from the high species specificity of astroviruses in general. Furthermore, HAstV-1-8 serotypes have similar tendencies to infect only select human and primate cells. To further investigate the possibility of a conserved receptor-binding site, the Inventors performed an alignment of the HAstV-1-8 capsid spike sequences (-35% homology) and mapped conserved residues onto the HAstV-1 spike structure (Fig. 7).
  • the Inventors identified two sites of surface-exposed, conserved and clustered amino acids that are the Inventors' initial receptorbinding site candidates. Interestingly, one of these receptor-binding sites lies on the side of the spike and may be inaccessible in the immature, uncleaved form of HAstV, but accessible in the mature, cleaved form of HAstV (Fig. 8, Fig. 14). The Inventors postulate that this could be a mechanism for HAstV cell exit, since HAstV does not induce lysis of human cells.
  • HAstV could have a glycan receptor.
  • Recombinant HAstV- 1 capsid spike was submitted for glycan microarray analyses and tested in replicates of six for binding to 611 different glycans at neutral and acidic pH. The Inventors found that HAstV capsid spike does not significantly binding to glycans, and only a few weak binding events to known sticky glycans was observed (Fig. 9).
  • HAstV capsid spike which alone binds strongly and specifically to the Caco-2 cell surface.
  • HAstV capsid spike which alone binds strongly and specifically to the Caco-2 cell surface.
  • HAstV capsid spike contains a receptor-binding site composed of conserved residues.
  • the novel fluorescence microscopy assay described herein suggests that EGFP-Spike specifically binds to the surface of Caco-2 cells (Fig. 6). By mutating candidate conserved receptor-binding site residues and test EGFP-Spike-mutants for the ability to bind Caco-2 cells one can generate mutations that do not induce misfolding and the Inventors have produced three EGFP-Spike-mutants in the lab that are soluble.
  • Fluorescence microscopy assay can be utilized with several additional controls, this includes test binding of EGFP-fused HAstV capsid shell domain (Fig. 2), which should not bind Caco-2 cells, and will further support the prominent role of HAstV spike in receptor- binding.
  • Fig. 2 One can further test binding of EGFP-fused turkey AstV capsid spike domain (Fig. 2), which should also not bind Caco-2 cells, as turkey AstV has a dramatically different structure with no conserved residues compared to HAstV. As such, turkey AstV, and other avian AstV, have a very different cell surface receptor compared HAstV.
  • the produced serum should contain HAstV capsid spike-specific antibodies that block EGFP-Spike binding to Caco-2 cells, and one can include fluorescent endosome stains in 24-hour time point samples to determine that EGFP-Spike does indeed co-localize with endosomes. Addition of inhibitors of endocytosis and endosome acidification that were recently found to inhibit HAstV infectivity can be utilized to further confirm cellular activity.
  • HAstV capsid spike binds to a proteinaceous cell surface receptor.
  • Far Western Blot assay which shows that EGFP-Spike specifically binds to discrete Caco-2 cell proteins (Fig. 10)
  • Immunoprecipitation studies could be performed with many variations, including pre-binding of EGFP-Spike to Caco-2 cells followed by detergent solubilization of the spike-receptor complex, or production of EGFP- Spike-coated beads for affinity pull-downs of receptor from lysates. While it is possible that detergents used to lyse Caco-2 cells could destroy the receptor's ability to bind HAstV spike, it appears that will not be the case, given the successful results of denaturing Far Western Blot assay.
  • N-terminal sequencing may facilitate identification as cell surface receptors usually are directed to the cell surface by a secretion signal, which is removed, leaving an unmodified N-terminus available for a successful Edman degradation reaction.
  • receptor candidates may be identified using a size range estimated in the Inventors' Far Western Blot assay.
  • HAstV capsid spike cell surface receptor candidates Having identified HAstV capsid spike cell surface receptor candidates, one can further validate the identity of the receptor by testing whether antibodies specific to a receptor candidate are able to block binding of EGFP-Spike to the Caco-2 cell surface. Additionally, siRNA knockdown of receptor candidates and test for decreased binding of EGFP-Spike to the Caco-2 cell surface may help to confirm receptor identity. Most interestingly, expression of a receptor candidate in cells that are found not to otherwise permit binding by EGFP- Spike, and then testing for EGFP-Spike binding would provide a strong indication of successful receptor identification. Further validation of the HAstV cell surface receptor can occur by testing for diminished binding to HAstV spike mutants.
  • the above results provide a major advancement in understanding how HAstV infects human cells.
  • the identity of the HAstV cell surface receptor(s) should provide insight into the reasons for the extraordinar cell type and species specificity of mammalian astroviruses, and this insight may bring new methodologies to propagate and study astrovirus in more convenient cell lines.
  • These studies will advance the field of virology by paving the way for virologists to test the role of the identified cell surface receptor in the broader context of HAstV infection in cells and animal models. As often happens when investigating virus-host interactions, these studies may also yield unexpected insight into the mechanisms of human cell surface proteins and their role in endocytosis after HAstV attachment.
  • HAstV capsid epitope for the HAstV neutralizing Monoclonal antibody PL-2 (MAb PL-2) one can test the immunogenicity of a HAstV capsid spike immunogen. It is suggested that the HAstV capsid spike domain elicits and binds HAstV- neutralizing antibodies that block receptor-binding.
  • HAstV capsid protein In terms of its structural domains, the shell, spike, and acidic domains, whose amino acid borders the Inventors determined theoretically using structural and bioinformatics tools (Figs. 3, 7). This approach eliminated the challenges in studying the HAstV in its multidomain, oligomerized, and proteolyzed form. Both the recombinant HAstV capsid shell and spike domains produced in E.
  • coli are soluble, folded, and retain antigenic properties as assessed by reactivity to anti -HAstV- 1 rabbit serum polyclonal antibodies (generated by HAstV- 1 virus immunization) in both Western Blot (linear epitopes) and ELISA (conformational epitopes preserved) (Fig. 3).
  • MAb PL-2 is reported to be a potent neutralizing antibody with high specificity for serotype HAstV-2 capsid.
  • Fig. 4A MAb PL-2 purified with immobilized Protein G beads followed by size-exclusion chromatography was evaluated.
  • MAbs bind to purified HAstV spike in an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Recombinant HAstV-2 spike bound strongly to pure MAb PL-2 by ELISA.
  • the Inventors found that MAb PL-2 did not bind HAstV-1 spike or shell domains (Fig. 4C, D).
  • Fig. 12 A To validate the structure of HAstV-2 spike, the Inventors determined its 1.9 A three-dimensional structure by X-ray crystallography and found that it folds as a dimer.
  • HAstV-2 spike dimer This test would reveal whether there was a single epitope at the interface of the HAstV-2 spike dimer, or whether there were two epitopes per HAstV-2 spike dimer.
  • the Inventors isolated the MAb PL-2 antigen-binding Fab fragments (Fab PL-2) with immobilized papain, Protein G bead removal of the Fc fragment, and tandem anion exchange and size-exclusion chromatography steps. The Inventors then mixed Fab PL- 2 and HAstV-2 spike, in ratios with excess Fab or excess spike. Samples were analyzed by size-exclusion chromatography and SDS-PAGE analyses (Fig. ⁇ ⁇ , ⁇ ).
  • the Inventors performed crystallization trials that produced initial needle crystals, which the Inventors optimized using crystal microseeding methods (Fig. 11C).
  • the Inventors solved the preliminary 1.9 A resolution crystal structure of Fab PL-2 fragment by X-ray crystallography (Fig. 12B). At this resolution, one can deduce de novo the amino acid sequence of -90% of the Fab, with high confidence at nearly every amino acid in the CDR loops, and mass spectrometry data has resulted in the de novo Fab PL-2 sequence.
  • the Fab PL-2 sequence allows generation endless recombinant MAb PL-2, Fab PL-2, or single-chain variable fragment (scFv) PL-2 such as soluble, folded scFv PL-2 in transiently-transfected Schneider 2 insect cells (Fig. 13).
  • MAb PL-2 binds strongly to recombinant HAstV-2 capsid spike domain.
  • MAb PL-2 was reported to recognize HAstV-2 capsid protein by immunoprecipitation or enzyme-linked immunosorbent assay (ELISA), but not by denaturing Western Blot, suggesting targeting of a conformational-dependent epitope.
  • ELISA enzyme-linked immunosorbent assay
  • Three- dimensional structural information may be required to identify the key molecular features governing the interaction between MAb PL-2 and HAstV-2 capsid spike.
  • X-ray crystallography can aid determination of the high resolution three-dimensional structure of the Fab PL-2 / HAstV-2 capsid spike domain complex (Fab/spike complex).
  • Fab/spike complex capsid spike domain complex
  • These studies require milligram amounts of Fab/spike complex that is highly pure, concentrated, and stoichiometrically homogenous. Purification of the Fab PL-2 and the HAstV-2 spike have led to the high-resolution structures of each sample (Fig. 12) while supporting optimized production of Fab/spike complex at the small-scale level (Fig. 11). Crystals of the Fab/spike complex are shown (Fig. 11C).
  • Crystallization of scFv PL-2 / HAstV-2 spike complex will be highly informative as trimmed glycosylation of scFv produced in insect cells, in addition to the more compact scFv fragment itself, will aid in crystallogenesis of the complex.
  • Recombinant dimeric HAstV capsid spike antigen will be an effective immunogen and elicit high levels of antibodies that block EGFP-spike receptor-binding activity.
  • the described results represent the first structural and mechanistic investigation into HAstV neutralization of HAstV. These studies will advance the field of virology by paving the way for virologists to test the effectiveness of the HAstV capsid spike vaccine in the broader context of protection from HAstV infection in cells and animal models. These studies will also provide new avenues for the development of antiviral therapeutics for immune- compromised patients with severe or persistent HAstV infection. Finally, these studies may stimulate studies testing the correlation between serum antibodies targeting HAstV capsid spike and protection from HAstV disease.
  • scFv single-chain variable fragment
  • the advantages of recombinant scFv include increased homogeneity and purity, increased yields, and in the case of recombinant scFv, a more compact molecule that may be advantageous for crystallogenesis.
  • the Inventors' lab currently has the necessary technology to produce recombinant antibody fragments in S2 insect cells.
  • the Inventors' "last-resort" alternative approach to characterize the site of MAb PL-2 neutralization is to produce singlepoint amino acid mutations on the HAstV-2 capsid spike surface and use ELISA to test mutant HAstV-2 capsid spike samples for reduced MAb PL-2 binding.
  • MAb PL-2 does not block GFP-spike attachment to Caco-2 cells, and the Inventors would interpret this result as MAb PL-2 neutralizing HAstV-2 at another point in vims entry following cell attachment, such as host membrane penetration or virus uncoating.
  • MAb PL-2 neutralizing HAstV-2 at another point in vims entry following cell attachment, such as host membrane penetration or virus uncoating.
  • serum contained no antibodies that block receptor-binding activity Antigens that bind specific antibodies do not necessarily elicit the same specific antibodies, and one hypothesis is that cathepsin processing of antigens in cells destroys the epitope.
  • An alternative strategy would be to identify and mutate cathepsin cleavage sites without destroying epitope, and then immunize again with the resulting mutant HAstV-1 spike antigen.
  • Another alternative strategy to enhance the production of neutralizing antibodies would be to boost vaccinated animals with a peptide containing part/all of the neutralizing epitope sequence.
  • HAstV capsid spike protein can indeed elicit neutralizing antibodies in mice, thereby providing strong evidence that the proposed vaccine provides a protective antibody response.
  • HAstV capsid spike is the main antigenic domain.
  • SDS- PAGe, Western Blot and ELISA detection demonstrate reactivity to anti-HAstV-1 rabbit serum polyclonal antibodies, or a-HAstV-1 polyclonal sera.
  • sequence divergent turkey AstV TAstV-2
  • mice immunized with recombinant HAstV-1 capsid spike develop a HAstV-1 -neutralizing antibody response.
  • intestinal caco-2 cell monolayers ae infected with HAstV-1 that had been previously incubated with serial dilutions of sera raised in mice to the HAstV capsid spike domain (Spikel) or to the complete purified HAstV-1 virus.
  • the cells were fixed, permeabilized, and incubated with rabbit anti-HAstV serum, followed by anti-rabbit IgG antibodies coupled to Alexa 488.
  • Infected cells were analyzed by fluorescence microscopy. The dilution of the sera is indicated and control virus was incubated with preimmune mouse serum.
  • HAstV capsid spike binds to caco-2 cells and binding is blocked by addition of scFv PL-2 antibody.
  • SDS-PAGE detection allowed measurement of labeled spike protein.
  • scFv PL-2 antibody in excess reduced detection of bound HAstV.
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

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Abstract

La présente invention concerne des procédés et des compositions associés au développement de solutions préventives pour résoudre le problème des maladies et des pathologies liées à l'astrovirus, telles que la diarrhée. Compte tenu de la taille et du traitement complexe de la protéine d'astrovirus, l'étude de ce virus est difficile. Il n'existe aucun vaccin contre l'astrovirus, ni aucune technologie associée aux vaccins classiques, telle que l'inactivation de virus ou l'atténuation du virus. Au lieu d'une approche centrée sur des sous-unités moléculaires importantes pour l'activité virale, une approche vaccinale centrée sur une sous-unité est susceptible de présenter peu d'effets secondaires et peut être produite facilement et de manière abordable. Des études biochimiques et structurales permettent d'établir des éléments nécessaires pour susciter une réponse immunitaire d'anticorps anti-astrovirus protecteurs et empêcher la possibilité d'une infection par un astrovirus.
PCT/US2016/021987 2015-03-13 2016-03-11 Vaccin composé de sous-unités pour la prévention d'une maladie causée par l'astrovirus WO2016149089A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111905100A (zh) * 2020-08-17 2020-11-10 山东信得科技股份有限公司 一种鹅星状病毒二价灭活疫苗和卵黄抗体及其制备方法
CN116970574A (zh) * 2023-06-26 2023-10-31 广东省农业科学院动物卫生研究所 一种鹅源波形蛋白的用途、促进增殖鹅星状病毒的方法及用途、疫苗
CN116970574B (zh) * 2023-06-26 2024-02-09 广东省农业科学院动物卫生研究所 一种鹅源波形蛋白的用途、促进增殖鹅星状病毒的方法及用途、疫苗

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