WO2016149089A1

WO2016149089A1 - Subunit vaccine for prevention of astrovirus disease

Info

Publication number: WO2016149089A1
Application number: PCT/US2016/021987
Authority: WO
Inventors: Rebecca DUBOIS
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2015-03-13
Filing date: 2016-03-11
Publication date: 2016-09-22
Anticipated expiration: 2017-09-13

Abstract

Described herein are methods and compositions related to developing preventative solutions to the problem of astrovirus-related disease and conditions, such as diarrhea. Given the size and complex processing of astrovirus protein, study of this virus is challenging. No astrovirus vaccine exists and conventional vaccine technologies such as virus inactivation or virus attenuation have not been published. By instead focusing on molecular subunits critical to viral activity, a subunit-focused vaccine approach is likely to have low side effects and can be produced easily and affordably. Structural and biochemical studies allow one to establish elements necessary to elicit a protective anti-astrovirus antibody immune response and prevent possibility of astrovirus infection.

Description

SUBUNIT VACCINE FOR PREVENTION OF ASTROVIRUS DISEASE

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under K22 AI 095369-01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

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.

BACKGROUND

Human astrovirus (HAstV) is a leading cause of viral diarrhea in young children. With an estimated 3.9 million cases of astrovirus diarrhea per year in the United States alone, worldwide incidence of HAstV in children with gastroenteritis ranges between 7-23%. HAstV is also attributed to chronic gastroenteritis in hospitalized or immune-compromised children as well as the elderly. There are no vaccines or antiviral therapeutics for astrovirus disease.

Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against HAstV and this suggests that antibodies developed by the adaptive immune response play an important role in controlling astrovirus infection. First, the demographics of infection and rarity of astrovirus infection in adults suggests that they have a protective adaptive immune response. In fact, over 70% of healthy adults have astrovirus antibodies. Finally, two astrovirus clinical studies with healthy volunteers found that those with more severe diarrheal disease had no astrovirus antibodies. Taken together, these data suggest that a vaccine that induces a productive antibody response will protect individuals from astrovirus infection.

Despite the above results, traditional methods for astrovirus vaccine production have not yet been successful. The challenges in traditional approaches, such as virus attenuation or inactivation, arise from the fact that astrovirus is extremely stable and resistant to inactivation by alcohols, bleach, detergents, heat treatment, and UV treatment. An alternative vaccine approach using a recombinant full-length astrovirus capsid protein vaccine in minks showed some protection. However, several observations suggest that this recombinant capsid vaccine does not contain the proper conformational epitope(s). Compounding these challenges is a fundamental gap in knowledge regarding the binding sites and mechanisms of action of neutralizing antibodies that block HAstV infection. If these challenges are surmountable, 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.

SUMMARY OF THE INVENTION

Described herein is a vaccine for protecting a mammal against a disease condition resulting from an astrovirus infection, including a subunit of an astrovirus and an adjuvant. In other embodiments, the subunit of an astrovirus includes a capsid protein. In other embodiments, the subunit of an astrovirus includes a capsid protein spike. In other embodiments, the capsid protein spike includes a receptor binding domain. In other embodiments, the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the disease condition includes diarrhea or gastroenteritis.

Further described herein is a method of administering a vaccine, wherein the vaccine is for protecting a mammal against a disease condition resulting from an astrovirus infection. In various embodiments, the vaccine includes subunit of and an adjuvant. In other embodiments, the subunit of an astrovirus includes a capsid protein. In other embodiments, the subunit of an astrovirus includes a capsid protein spike. In other embodiments, the capsid protein spike includes a receptor binding domain. In other embodiments, the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the disease condition includes diarrhea or gastroenteritis.

Also described herein is a composition including cells capable of producing one or more astrovirus proteins. In other embodiments, the one or more astrovirus proteins comprise a capsid protein. In other embodiments, the capsid protein includes a capsid protein spike. In other embodiments, the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the cells are eukaryotic. In other embodiments, the cells are prokaryotic. In other embodiments, the astrovirus protein includes a histidine tag. In other embodiments, the histidine tag includes a C-terminal or N-terminal tag.

Also described herein is method of producing one or more astrovirus proteins using includes a composition including cells capable of producing one or more astrovirus proteins. In other embodiments, the one or more astrovirus proteins comprise a capsid protein. In other embodiments, the capsid protein includes a capsid protein spike. In other embodiments, the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the cells are eukaryotic. In other embodiments, the cells are prokaryotic. In other embodiments, the astrovirus protein includes a histidine tag. In other embodiments, the histidine tag includes a C-terminal or N-terminal tag.

Further described herein is a method of inducing an immune response against astrovirus including administering one or more astrovirus proteins to a mammal. In other embodiments, 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. In other embodiments, inducing an immune response includes production of one or more antibodies specific to one or more astroviruses. In other embodiments, the one or more antibodies comprise a monoclonal antibody. In other embodiments, the one or more antibodies comprise a polyclonal antibody.

BRIEF DESCRIPTION OF FIGURES

Figure 1. Astroviruses infection of mammals and birds.

Figure 2. Astrovirus virion and capsid protein processing. (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.

Figure 3. Both HAstV capsid shell and spike domains are antigenic. (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.

Figure 4. Neutralizing MAb PL-2 binds with high specificity to HAstV-2 spike. (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.

Figure 5. High-resolution crystal structures of recombinant HAstV-1 and HAstV-2 capsid spike. (Fig. 5A) HAstV-1. (Fig. 5B) HAstV-2.

Figure 6. HAstV capsid spike binds to Caco-2 cells in a specific manner. Caco-2 cells were treated with 5 to 10 μΜ 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.

Figure 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. Conserved amino acids are colored red, and homologous residues are pink. Two patches of conserved residues are circled in blue or red.

Figure 8. 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.

Figure 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.

Figure 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 μg of Caco-2 whole cell lysate was loaded onto each lane as indicated above. BSA-blocked nitrocellulose membranes were incubated with 5 μΜ of EGFP-Spike or 5μΜ 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.

Figure 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.

Figure 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.

Figure 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.

Figure 14. Astrovirus virion and capsid protein domains. (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-

1 virion T=3 icosahedral capsid.

Figure 15. 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.

Figure 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. At 18 h postinfection 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.

Figure 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).

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al, Remington: The Science and Practice of Pharmacy 22^nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al, Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3^rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March 's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7^th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3^rd ed., Wiley -Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2^nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al, Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

As described, human astrovirus (HAstV) 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. Relevant to the food industry, 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.

Astrovirus particles are composed of a small, positive-sense, single-stranded RNA genome surrounded by a ~35nm T=3 icosahedral capsid protein shell. 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 . One study isolated three neutralizing MAbs, each of which block virus attachment to cells (receptor- binding) MAbs 5B7, 7C2, and 3B2. Another study isolated a neutralizing MAb, called PL-2, that bound mature HAstV capsid proteolytic fragments. These fragments were later mapped to the capsid spike domain. Interestingly, MAb PL-2 only recognizes HAstV by immunoprecipitation or enzyme-linked immunosorbent assay (ELISA), but not by denaturing Western Blot, strongly suggesting that the neutralizing MAb PL-2 targets a conformational - dependent HAstV epitope.

Despite the above results, traditional methods for astrovirus vaccine production have not yet been successful. There may be a variety of reasons for why such traditional methods of HAstV vaccine generation by virus attenuation or virus inactivation methods have not been established. First, only in the last decade with advancements in molecular surveillance technologies has HAstV been recognized as a leading cause of viral gastroenteritis. Thus, HAstV is an understudied pathogen. Second, 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. Third, HAstV virions are highly stable and resistant to chemical and UV inactivation, suggesting that virus inactivation may be challenging. Fourth, while 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. Finally, 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.

Certain non-traditional approaches to astrovirus vaccine production using recombinant full-length astrovirus capsid protein as a vaccine antigen have been published. It has been reported that one can produce purified recombinant mink astrovirus capsid protein from stably-transfected baby hamster BHK-21 cells. This purified astrovirus capsid protein can be used to vaccinate mink and showed partial protection from mink astrovirus disease. Alternatively, it has been reported that one can produce recombinant chicken astrovirus capsid protein using bacuolvirus expression in insect cells, with purified protein being used to vaccinate chickens. Protection from runting-stunting syndrome in chickens was observed, but again, such protection was only partial. It is suggested that only partial protection from disease is observed under these circumstances because the vaccine does not sufficiently mimic the astrovirus virion. Hence, the described approaches may not be fully eliciting protective antibodies that recognize and neutralize the virus. Several biochemical observations regarding the recombinant full-length astrovirus capsid antigen vaccines support the notion that the capsid protein does not form virus-like particles and does not undergo proteolytic processing by host cell proteases.

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. There are considerable difficulties in studying the HAstV in its multidomain, oligomerized, and proteolyzed form. 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. Despite this apparent complexity, it is suggested that the HAstV capsid spike composes a RBD. First, 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. Second, 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. Finally, 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.

As the proposed solution focuses on developing a vaccine approach focused on subunits as an immunogen. First the emerging field of structure-based vaccine design allows for the development of improved immunogens that could not otherwise be obtained by traditional methods. Current structural and antigenic data support the notion that specific vaccine antigens can mimic the actual HAstV virus surface. Second, an affordable, simple, and scalable strategy can be developed to produce a HAstV subunit vaccine to establish the possibility of global vaccination. This strategy eliminates the previous challenges and limitations in producing the entire functional and antigenic form of the HAstV capsid protein. As described, 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. Third, vaccine production strategy uses standard bacterial cell lines that are already used in therapeutic protein production, thus low side effects are expected.

Without being bound by any particular theory, it is suggested that the described approach for developing a vaccine can provide a preventative solution to the problem of astrovirus-related disease such as diarrhea. Purified 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. Based on the reports described herein, 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. To overcome these barriers, 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. Using an arsenal of integrated methodologies, 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 affordable, simple, and scalable strategy the Inventors have developed to produce HAstV capsid spike immunogen hints at the possibility of global vaccination. Furthermore, 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. Finally, by studying the mechanistic framework that underlies HAstV receptor-binding and HAstV neutralization, 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.

Described herein are methods and compositions for development of a safe and effective astrovirus vaccine. By 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. One can then test one or more astrovirus capsid protein subunit antigens for the ability to elicit protective neutralizing antibodies in animals. By visualizing in molecular detail how viruses enter and replicate in human cells, one can use this information to develop new vaccines and antiviral therapeutics.

Specifically, 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. For example, one can target 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.

By determining the structural basis of astrovirus neutralization and delineating the sites of vulnerability on the astrovirus capsid protein, 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.

For example, X-ray crystallographic mapping to understand the molecular basis for astrovirus neutralization. As described, neutralizing MAb PL-2 are thought to recognize a conformation-dependent epitope on the -26 and ~29kD fragments of the astrovirus capsid protein. Subsequent studies have shown that these fragments compose the region of the capsid that forms dimeric spike projections on the virus surface. Using methods further described herein, the Inventors have successfully produced in E. coli recombinant spike domain from HAstV-2, the strain used to make MAb PL-2. Importantly, crystallized HAstV- 2 spike protein confirms correct folding and resolution of its preliminary atomic structure. Similarly, the Inventors have produced HAstV-1 capsid spike, crystallized, and solved the structure of the capsid spike. One can also explore binding of antibodies such as MAb PL-2 to HAstV capsid spike, such as purified HAstV-2 spike, in an enzyme-linked immunosorbent assay (ELISA). Such technique helping elucidate binding interactions that ultimately be involved in acquisition of immunity.

Extending the above, one can produce 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. In this regard, 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. Instead, one can produce and purified the antigen-binding Fab fragments of MAb PL-2 using papain resin and Protein A affinity removal of the Fc fragment. Following purification of the MAb Fab, binding stoichiometry and structure of the Fab PL-2/HAstV-2 spike complex is of great interest. Specifically, understanding whether 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.

Additionally, one can complement structural results by biochemically characterizing astrovirus sites of vulnerability and neutralization. For example, by producing point mutants of HAstV-2 capsid spike for MAb binding studies allowing one to confirm sites predicted by the Fab PL-2/HAstV-2 spike complex structure to be important for binding. Alternatively, existing HAstV spike structures will provide for choice of point mutations in highly conserved surface residues that are Inventors predicted to be sites of vulnerability and neutralization. Thereafter, test binding of MAb PL-2 to HAstV-2 capsid spike mutants using ELISA can serve to ascertain whether single point mutants, combined double and triple mutants contribute to binding complex structures. To assess binding in a more quantitative manner, one can use 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. One can test binding of HAstV spike mutants and HAstV-2 spike in the presence of MAb PL-2 and also use this assay to probe the identity of HAstV host cell receptor. 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.

Additional studies involve characterizing antigenicity and vaccine potential of astrovirus capsid antigens. For example, 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. When immunized with 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.

Described herein is a vaccine for protecting a mammal against a disease condition resulting from an astrovirus infection, including a subunit of an astrovirus and an adjuvant. In other embodiments, the subunit of an astrovirus includes a capsid protein. In other embodiments, the subunit of an astrovirus includes a capsid protein spike. In other embodiments, 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. In other embodiments, the capsid protein spike includes a receptor binding domain. In other embodiments, the astrovirus includes human astrovirus (HAstV)- 1, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc. In other embodiments, the disease condition includes diarrhea or gastroenteritis.

Further described herein is a method of administering a vaccine, wherein the vaccine is for protecting a mammal against a disease condition resulting from an astrovirus infection. In various embodiments, the vaccine includes subunit of and an adjuvant. In other embodiments, the subunit of an astrovirus includes a capsid protein. In other embodiments, the subunit of an astrovirus includes a capsid protein spike. In other embodiments, 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. In other embodiments, the capsid protein spike includes a receptor binding domain. In other embodiments, the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc. In other embodiments, the disease condition includes diarrhea or gastroenteritis. In other embodiments, 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.

Also described herein is a composition including cells capable of producing one or more astrovirus proteins. In other embodiments, the one or more astrovirus proteins comprise a capsid protein. In other embodiments, the capsid protein includes a capsid protein spike. In other embodiments, 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. In other embodiments, the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc. In other embodiments, the cells are eukaryotic. In other embodiments, the cells are prokaryotic. In other embodiments, the cells are mink or chicken cells. In other embodiments, the cells are insect cells. In other embodiments, the astrovirus protein includes a histidine tag. In other embodiments, the histidine tag includes a C-terminal or N-terminal tag. In other embodiments, the astrovirus protein includes a fluorescent label such as green fluorescent protein (GFP).

Also described herein is method of producing one or more astrovirus proteins using includes a composition including cells capable of producing one or more astrovirus proteins. In other embodiments, the one or more astrovirus proteins comprise a capsid protein. In other embodiments, the capsid protein includes a capsid protein spike. In other embodiments, the astrovirus includes human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8. In other embodiments, the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc. In other embodiments, the cells are eukaryotic. In other embodiments, the cells are prokaryotic. In other embodiments, the astrovirus protein includes a histidine tag. In other embodiments, the histidine tag includes a C-terminal or N-terminal tag. In other embodiments, the astrovirus protein includes a fluorescent label such as green fluorescent protein (GFP).

Further described herein is a method of inducing an immune response against astrovirus including administering one or more astrovirus proteins to a mammal. In other embodiments, 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. In other embodiments, the astrovirus includes astrovirus strains in poultry, swine, cows, dogs, cats, mice, rabbits, mink, etc. In other embodiments, inducing an immune response includes production of one or more antibodies specific to one or more astroviruses. In other embodiments, the one or more antibodies comprise a monoclonal antibody. In other embodiments, the one or more antibodies comprise a polyclonal antibody.

This described methods and compositions directly impact human health by providing fundamental, atomic resolution structural and mechanistic insight into the HAstV capsid protein. These molecular insights will significantly advance the field of astrovirus biology and provide a foundation for the development of novel therapeutic strategies to prevent and treat astrovirus gastroenteritis.

Example 1

Study design

As described, the current studies focus on producing an astrovirus subunit vaccine including a recombinant astrovirus capsid "spike" domain. 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). Mature astrovirus particles are composed of a small, positive-sense, single-stranded RNA genome surrounded by a ~35nm T=3 icosahedral capsid protein shell projecting 30 knob-like projections called spikes (Fig. 2A), as shown in reported crystal structures of the human and avian astrovirus spikes (Fig. 2B).

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. By engineering the protein construct with a removable C- terminal 10-histidine-tag, 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).

Example 2

Structure validation

Only two reports published over twenty years ago describe the isolation of neutralizing monoclonal antibodies (MAbs) targeting HAstV, one of which is MAb PL-2 produced in mouse ascites fluid. MAb PL-2 is reported to be a potent neutralizing antibody with high specificity for serotype HAstV-2. To further investigate the mechanism of MAb PL-2, the Inventors purified MAb PL-2 with immobilized Protein G beads followed by size- exclusion chromatography (Fig. 4A). The Inventors then tested whether the MAbs bind to purified HAstV spike in an enzyme-linked immunosorbent assay (ELISA). For this purpose, 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).

To validate the structures of HAstV- 1 and HAstV-2 capsid spike proteins produced in the Inventors' lab, the Inventors determined their high-resolution (0.9 and 1.9 A) three- dimensional structures by X-ray crystallography (Fig. 5). The Inventors found that the Inventors' recombinant spikes proteins have dimeric structures very similar to that from HAstV-8. Example 3

Capsid Ststructure as receptor binding domain

Finally, to understand the mechanism of HAstV neutralization by MAb PL-2, the Inventors asked whether the HAstV capsid spike domain is the virus's receptor-binding (i.e. cell attachment) domain. Several observations support the suggestion that the HAstV capsid spike composes a receptor-binding domain (RBD). First, 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. Second, 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. Finally, 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.

To directly test the role of the HAstV capsid spike in attachment to human cells, the Inventors have developed a novel fluorescence microscopy assay to directly visualize HAstV capsid spike attachment and endocytosis into cells. First, 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). For a control, the Inventors also produced EGFP alone. The 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. Moreover, at the 24-hour time point, 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. Example 4

Preliminary results

Taken together, the Inventors' preliminary data show that 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. Further, recombinant HAstV capsid spike is a HAstV receptor-binding domain. Also, HAstV-neutralizing antibodies binding the HAstV capsid spike may function by blocking virus attachment to human cells

The Inventors' data suggest that a HAstV subunit vaccine composed of the spike domain will elicit anti -HAstV neutralizing antibodies. Extending these results, it is suggested that 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). First, the Inventors will use rabbit antibody production with standard immunization protocols to test the immunogenic properties of HAstV- 1 capsid spike. 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. Such results are directed at human astrovirus, but 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.

Example 5

Identify the HAstV receptor-binding site and the HAstV cell surface receptor (s) HAstV attaches to human cells via specific interactions between a conserved HAstV capsid spike receptor-binding site and a cell surface receptor. To test this, 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. Finally, the Inventors demonstrate that 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).

Example 6

Characterize a neutralizing epitope and immunogenicity of the HAstV capsid spike

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.

Example 7

HAstV capsid domain, the spike

By focusing on a single HAstV capsid domain, the spike, and not the entire HAstV capsid protein, for example, one can eliminate the previous challenges and limitations in producing the entire functional and antigenic form of the HAstV capsid protein. Recombinant 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. Although 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.

For the aforementioned reasons, structure-based vaccine design allows for the design of improved immunogens that could not be obtained by traditional methods. Vaccination of animal with a HAstV immunogen that the Inventors have structurally designed and validated to correctly present a conformational neutralizing epitope. 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.

Example 8

Identify the HAstV receptor-binding site and the HAstV cell surface receptor(s)

The molecular mechanisms by which HAstV attaches to human cells, and the identity of the cell surface receptor(s), remain elusive. Of key interest is identifying the receptor- binding site on HAstV capsid protein and uncovering the identity of the HAstV cell surface receptor(s). It is suggested that HAstV attaches to human cells via specific interactions between a conserved HAstV capsid spike receptor binding site and a cell surface receptor.

As described, mature HAstV particles are composed of a small, positive-sense, single stranded RNA genome surrounded by a ~35nm T=3 icosahedral capsid protein shell projecting 30 knob-like projections called spikes (Fig. 2 A). 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.

Example 9

HAstV serotype 1 (HAstV- 1) capsid spike domain as a recombinant protein in E. coli To begin to test the role of the HAstV capsid spike in attachment to human cells, 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.

It is suggested that 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). On each protomer of the dimer, 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.

Example 10

Characterization of the HAstV cell surface receptor

It is suggested that 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).

While many recombinant viral glycan-binding proteins bind poorly to the microarray because they lack the avidity effect of multivalency on the virus surface, it is suggested that this is not the case for HAstV capsid spike, which alone binds strongly and specifically to the Caco-2 cell surface. One can then test for binding by HAstV capsid spike to a proteinaceous receptor by developing a Far Western Blot assay. In this assay, varying amounts of Caco-2 cell ly sates are separated by SDS-PAGE and blotted to a nitrocellulose membrane. After blocking, one can incubate membranes with purified EGFP or EGFP-Spike (Fig. 9A), wash the membranes, and probe for specific binding using a HRP-conjugated antibody recognizing the histidine purification tag at the N-termini of the EGFP proteins. This approach establishes that HAstV capsid spike binds specifically to several discrete bands between 100-250kD, suggestive of binding to a proteinaceous Caco-2 cell surface receptor (Fig. 10). Example 11

Structurally and biochemically characterize the HAstV capsid spike receptor-binding site It is suggested that that 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. 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. One can also test anti -HAstV- 1 rabbit serum polyclonal antibodies (generated by HAstV- 1 virus immunization) for the ability to block EGFP-Spike binding to Caco-2 cells. 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.

One can also test other non-primate cell lines for EGFP-Spike binding to the cell surface and endocytosis. It is suggested that EGFP-Spike will not bind to non-primate cells because they lack the HAstV receptor. This can be confirmed by testing several cell lines that are non-permissive to HAstV infection, including MDCK, CHO, and BHK-21 cells. Of particular interest are transformable cell lines, which could be used to validate candidate receptors. Together, these controls further validate the fluorescence microscopy assay, providing a direct method to probe and identify amino acids involved in HAstV capsid spike receptor-binding activity. Example 12

Biochemically identify HAstV capsid spike cell surface receptor candidate(s)

It is suggested that HAstV capsid spike binds to a proteinaceous cell surface receptor. Using the described Far Western Blot assay, which shows that EGFP-Spike specifically binds to discrete Caco-2 cell proteins (Fig. 10), one can further extend these results by biochemical fractionation of Caco-2 cell lysates, including cell membrane isolation, ammonium sulfate precipitation, ion exchange chromatography, and size- exclusion chromatography in combination with the Far Western Blot assay to test which fractions contain protein bands bound specifically by EGFP-Spike but not EGFP. In parallel studies, one can rely on immunoprecipitation experiments to specifically isolate HAstV receptor(s) from detergent- solubilized Caco-2 cell lysates. 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.

Finally, after isolating a HAstV receptor candidate sample to reasonable purity, one can rely on mass spectrometry to identify proteins in the sample. In combination with mass spectrometry, 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. In addition to these protein identification technologies, receptor candidates may be identified using a size range estimated in the Inventors' Far Western Blot assay.

Example 13

Biochemically and genetically validate the HAstV capsid spike cell surface receptor (s)

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 exquisite 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.

Example 14

By characterizing the 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.

To identify the sites of the HAstV capsid epitopes, one may visualize the 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).

As described, only two reports published over twenty years ago describe the isolation of neutralizing MAbs targeting HAstV including MAb PL-2 in mouse ascites fluid. MAb PL- 2 is reported to be a potent neutralizing antibody with high specificity for serotype HAstV-2 capsid.

To further investigate the mechanism of MAb PL-2, MAb PL-2 purified with immobilized Protein G beads followed by size-exclusion chromatography was evaluated (Fig. 4A). Of interest was whether MAbs bind to purified HAstV spike in an enzyme-linked immunosorbent assay (ELISA). Recombinant HAstV-2 spike bound strongly to pure MAb PL-2 by ELISA. 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, D). 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 (Fig. 12 A).

The Inventors then wanted to test the stoichiometry of MAb PL-2 binding of the

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. To test this, 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. Ι ΙΑ,Β). The Inventors found that having excess Fab produced a homogenous peak of Fab PL-2 / HAstV-2 capsid spike domain complex (Fab/spike complex), whereas excess spike produced heterogeneous doublet peaks of complex. The Inventors' data support a 2:2 Fab:Spike binding stoichiometry model. This suggests that the Fab PL-2 binding sites (epitopes) are sufficiently distal on the HAstV-2 spike dimer to allow two Fab fragments to bind. A model for the Inventors' results is illustrated in Fig. 1 ID.

To further characterize the Fab PL-2, 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).

Example 15

Structurally characterize the site of HAstV neutralization by monoclonal antibody PL-2

Neutralizing antibody 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. 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). 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.

Example 16

Biochemically and mechanistically characterize the HAstV capsid spike site of neutralization Of great interesting is understanding the mechanism of neutralization by confirming a cellular mechanisms by which MAb PL-2 binds at or near a receptor-binding site on the spike domain and neutralizes HAstV by preventing attachment to human cells. First, the Inventors will test in the Inventors' fluorescence microscopy receptor-binding assay (Fig. 6) the ability of MAb PL-2 to block attachment of EGFP-HAstV-2 spike will confirm if MAb PL-2 does indeed block the HAstV-2 spike receptor-binding activity. Further, production of single- point mutations of HAstV-2 capsid spike in residues found to interact with the Fab PL-2 in the complex structure will confirm if single-point mutants of EGFP-HAstV-2 spike can attach to Caco-2 cells. Identification of amino acids involved in HAstV receptor-binding, mutants, strain-specific amino acids in the interaction between MAb PL-2 and HAstV-2 spike are all likely to contribute to binding activity and specificity. For example, of great interest is understanding if single-point mutants of HAstV-2 capsid spike have decreased binding to MAb PL-2. One can first test binding of HAstV-2 spike mutants to MAb PL-2 using ELISA and assess binding more quantitatively, by using BIAcore surface plasmon resonance to measure binding on- and off-rates and calculate dissociation constants. These studies will extend the Inventors' understanding of the key amino acids involved in MAb PL-2 neutralization. Example 17

Characterize immunogenicity of recombinant dimeric HAstV capsid spike immunogen 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.

Example 18

Potential technical challenges

An obvious pitfall would be the inability to obtain high-quality crystals for the high- resolution structure of the Fab/spike complex. The Inventors have extensive experience in alternative pathways to crystallization, including optimizing sample purity/homogeneity/concentration/pH/salt content, binding complex to a second noncompeting Fab fragment (e.g., a second, non-competing, non-neutralizing MAb), chemical crosslinking, or using crystal seeds from spike alone or Fab alone to stimulate Fab/spike complex crystal growth. One explanation for crystallization difficulties might be slight heterogeneity in the Fab PL-2 sample due to alternative papain cleavage sites during MAb proteolysis or heterogeneous glycosylation. The Inventors are pursuing an alternative approach to engineer recombinant single-chain variable fragment (scFv) antibody. 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.

Alternatively, if 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. Further, in the event of low/no rabbit serum antibody titers one can attempt to increase titers using larger amounts of antigen or immunizing with adjuvants. Another potential pitfall would be if 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.

Example 19

Immunization with recombinant HAstV-1 capsid spike protein elicits neutralizing antibodies in mice

Using the aforementioned techniques, it is demonstrated that immunization with recombinant HAstV-1 capsid spike protein can indeed elicit neutralizing antibodies in mice, thereby providing strong evidence that the proposed vaccine provides a protective antibody response. As shown in Fig. 15, 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. By contrast, little or no cross- reactivity is observed with sequence divergent turkey AstV (TAstV-2) capsid domains. Further, as shown in Fig. 16, mice immunized with recombinant HAstV-1 capsid spike develop a HAstV-1 -neutralizing antibody response. Here, 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. At 18 h post-infection 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.

Extending the above results, it is shown in Fig. 17 that 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. Importantly, the presence of scFv PL-2 antibody in excess reduced detection of bound HAstV. These successful results indicate one could utilize mouse serum antibodies to neutralize other strains besides HAstV-1. Given conservation across astroviruses, the above proof-of-principle further indicates strong possibility of broad-protection vaccine as prepared by combining recombinant spikes from different HAstV strains. The Inventors envision four major summaries of the above results: (1) engineering of a subunit vaccine to elicit broadly neutralizing antibodies targeting HAstV-1-8 (2) development of a therapeutic humanized PL-2 antibody to treat patients with chronic or severe HAstV disease, (3) development of better diagnostics for HAstV, and (4) development of high-throughput screens for small molecules that block the receptor-binding site of the HAstV spike.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof. Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the methods for preventing or treating osteoporotic patients and related conditions, such as fractures, methods of isolating or modifying astrovirus protein used in the described techniques, compositions of therapeutic agents and/or targeting astrovirus and components thereof generated by the aforementioned techniques, techniques and composition and use of solutions used therein, and the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, 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.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventor for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.

Claims

THE CLAIMS

1. A vaccine for protecting a mammal against a disease condition resulting from an astrovirus infection, comprising:

a subunit of an astrovirus; and

an adjuvant.

2. The vaccine of claim 1, wherein the subunit of an astrovirus comprises a capsid protein.

3. The vaccine of claim 2, wherein the subunit of an astrovirus comprises a capsid protein spike.

4. The vaccine of claim 3, wherein the capsid protein spike comprises a receptor binding domain.

5. The vaccine of claim 1, wherein the astrovirus comprises human astrovirus (HAstV)-l, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.

6. The vaccine of claim 1, wherein the disease condition comprises diarrhea or gastroenteritis.

7. A method of administering the vaccine of claim 1.

8. A composition comprising cells capable of producing one or more astrovirus proteins.

9. The composition of claim 8, wherein the one or more astrovirus proteins comprise a capsid protein.

10. The composition of claim 9, wherein the capsid protein comprises a capsid protein spike.

11. The composition of claim 8, wherein the astrovirus comprises human astrovirus (HAstV)- 1, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV-7, or HAstV-8.

12. The composition of claim 8, wherein the cells are eukaryotic.

13. The composition of claim 8, wherein the cells are prokaryotic.

14. The composition of claim 8, wherein the astrovirus protein comprises a histidine tag.

15. The composition of claim 14, wherein the histidine tag comprises a C-terminal or N- terminal tag.

16. A method of producing one or more astrovirus proteins using the composition of claim 8.

17. A method of inducing an immune response against astrovirus comprising:

administering one or more astrovirus proteins to a mammal.

18. The method of claim 17, wherein 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.

19. The method of claim 17, wherein inducing an immune response comprises production of one or more antibodies specific to one or more astroviruses.

20. The method of claim 19, wherein the one or more antibodies comprise a monoclonal antibody.

21. The method of claim 20, wherein the one or more antibodies comprise a polyclonal antibody.