Patent Application
20240316176
A Sequence Listing submitted as an XML file is hereby incorporated by reference. The name of the XML file for the Sequence Listing is 2017-0103-SL.xml, the date of the creation of the XML file is Oct. 18, 2023, and the size of the ASCII text file is 69,632 bytes.
RVs cause severe acute gastroenteritis primarily in infants and young children, leading to ˜200,000 deaths, 2.3 million hospitalizations, and 24 million outpatient visits among children younger than 5 years of age globally each year [25-27]. The two current RV vaccines, RotaTeq (Merck) and Rotarix (GlaxoSmithKline, GSK), are effective in protecting children from severe RV cases in many developed countries [28, 29]. However, they have not shown satisfactory efficacies in most developing countries [30-32] in Africa and Asia, where most infection, morbidity, and mortality of RV occur and thus the RV vaccines are mostly needed.
Disclosed herein are vaccine compositions, in particular, polyvalent icosahedral compositions for antigen presentation. The disclosed compositions may contain an S particle made up of recombinant fusion proteins. The recombinant fusion proteins may include a norovirus (NoV) S domain protein, a linker protein domain operatively connected to the norovirus S domain protein, and an antigen protein domain operatively connected to said linker.
This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
As used herein, the term “antigen” may be used interchangeably with the terms “immunogen” and “immunogenic antigen”, as defined below. Technically speaking, an antigen is a substance that is able to combine with the products of an immune response once they are made, but is not necessarily able to induce an immune response (i.e. while all immunogens are antigens, the reverse is not true); however, the antigens that are discussed herein as the subject of the present invention are assumed to be immunogenic antigens, even when referred to as antigens.
The term “fusion protein” means a protein created through translation of a fusion gene, resulting in a single polypeptide with functional properties derived from each of the original proteins.
The term “immunity” means the state of having sufficient biological defenses to avoid infection, disease, or other biological invasion by a disease-causing organism.
The term “immunogenicity” means the ability of an immunogen to elicit a humoral and/or cell-mediated immune response.
The terms “immunogen” and “immunogenic antigen” mean a specific type of antigen that is able to induce or provoke an adaptive immune response in the form of the production of one or more antibodies.
The terms “immunogenic response” and “immune response” mean an alteration in the reactivity of an organisms' immune system in response to an immunogen. This can involve antibody production, induction of cell-mediated immunity, complement activation or development of acquired immunity or immunological tolerance to a certain disease or pathogen.
The terms “immunization” and “vaccination” mean the deliberate induction of an immune response, and involve effective manipulation of the immune system's natural specificity, as well as its inducibility. The principle behind immunization is to introduce an antigen, derived from a disease-causing organism, which stimulates the immune system to develop protective immunity against that organism, but wherein the antigen itself does not cause the pathogenic effects of that organism.
The term “infection” means the invasion of an animal or plant host's body tissues by a pathogen, as well as the multiplication of the pathogen within the body and the body's reaction to the pathogen and any toxins that it may produce.
The terms “Norovirus,” “NoV”, “Norwalk-like virus,” or “NLV” refer to any virus of the Norovirus genus in the Calicivirus family, and includes, without limitation, the following: Norwalk Virus (“NV”), MOH, Mexico, VA 207, VA 387, 02-1419, C59, VA 115, Hawaii, Snow Mountain, Hillington, Toronto, Leeds, Amsterdam, Idaho Falls, Lordsdale, Grimsby, Southampton, Desert Shield, Birmingham, and White Rivercap. NoVs cause acute gastroenteritis in humans.
As used herein, the letter “S” means “S domain” when used in the context of the described particles, for example, in S69A/58C/140C-VP8, which means the S-VP8 protein with 69A/58C/140C mutations. In other aspects, the nomenclature used may be, for example, S with “69A/58C/140C” denoted as a superscript.
The term “vaccine” means a biological preparation or composition that improves immunity to a particular disease. Vaccines are examples of immunogenic antigens intentionally administered to induce an immune response in the recipient.
The terms “multivalent vaccine” and “polyvalent vaccine” mean a vaccine designed to immunize against two or more strains of the same microorganism (such as NoV), or against two or more different microorganisms.
Noroviruses (NoVs) are members the Norovirus genus in the family Caliciviridae, causing epidemic acute gastroenteritis in humans with significant morbidity and mortality [4, 5]. Structurally, NoV virions are encapsulated by a protein capsid that is composed of a single major structural protein, the capsid protein or viral protein 1 (VP1). The crystal structures of NoV capsids revealed that NoV VP1 contains two principle domains, the N-terminal shell (S) and the C-terminal protruding (P) domains, linked by a short hinge [6]. The S domain builds the interior, icosahedral shell supporting the basic scaffold of a NoV virion, while the P domain constitutes the dimeric protrusions [7-10] to stabilize NoV capsid and recognize cell surface glycans as the host attachment factors or receptors [11-14].
In vitro expression of full-length NoV VP1 via a eukaryotic system resulted in self-formation of 180-valent virus-like particles (VLPs) that are structurally and antigenically similar to the authentic viral capsids [6, 15], while production of the P domain via the E. coli system formed P dimers that are structurally indistinguishable from those of NoV capsid [7-11, 16-19]. In addition, generation of modified NoV P domains assembled into different higher order particles or complexes, including the 12-valent small P particles [20], the 24-valent P particles [21, 22], and the 36-valent P complexes [23].
Unlike the P domain, the S domain has been less studied, although “thin layer” S particles were reported through expression of the S domain in the baculovirus/insect cell system [11, 24], likely equivalent to the 180-valent shells of NoV capsids. In this study, Applicant developed a new technology to produce unified, 60-valent S particles, referred as S60 particles, via the simple E. coli system and applied them as a multifunctional vaccine platform for antigen presentation for subunit vaccine development against rotavirus (RV) and other pathogens.
RVs cause severe acute gastroenteritis primarily in infants and young children, leading to ˜200,000 deaths, 2.3 million hospitalizations, and 24 million outpatient visits among children younger than 5 years of age globally each year [25-27]. The two current RV vaccines, RotaTeq (Merck) and Rotarix (GlaxoSmithKline, GSK), are effective in protecting children from severe RV cases in many developed countries [28, 29]. However, they have not shown satisfactory efficacies in most developing countries [30-32] in Africa and Asia, where most infection, morbidity, and mortality of RV occur and thus the RV vaccines are mostly needed. Applicant's recent studies suggested that the low RV vaccine efficacy in the developing countries could be due to mismatched P types of the vaccines with the changing predominant RV P types in the middle- and low-income nations [33, 34]. In addition, both current live attenuated vaccines remain costly and the replications of vaccine RVs in intestine after oral administration may be the cause of the increased risk of intussusception in vaccinated children [35-41]. Thus, new generation of RV vaccines that can overcome the mentioned limitations of the two current RV live vaccine are warranted.
RV P types are determined by viral protein 4 (VP4) that constitutes the spike proteins of a RV virion. Structurally each spike protein contains two major parts, the stalk formed by VP5 and the distal head built by VP8 [42]. VP5 and VP8 are cleavage products of VP4 by a trypsin. The VP8 is responsible for interaction with RV host attachment factor or receptors that are a group of cell surface glycans, including histo-blood group antigens (HBGAs) [33, 43-45]. Previously studies have shown that VP8 antigens elicit neutralizing antibodies that inhibit RV infection and replication in culture cells and protected immunized mice from RV infection [46, 47], and therefore, the VP8 antigen is an important vaccine target against RVs [46-49].
However, many defined neutralizing antigens, including RV VP8, face a common problem of low immunogenicity for non-replicating vaccine development, due to their small sizes with low valences. This problem can be solved via fusion or conjugation of the antigens to a large, polyvalent protein platform for enhanced immunogenicity. In this study, Applicant has provided solid evidence supporting significantly enhanced immunogenicity of the RV VP8 antigens after displayed by the NoV S60 particles as an effective vaccine platform. Applicant's data indicates that the S60-VP8 particle can be easily produced, stable, and highly immunogenic toward the displayed RV VP8 antigen, and thus is a promising subunit vaccine against RV infection.
Homotypic interactions of viral capsid proteins are common, driving viral capsid self-formation. By taking advantage of such interactions of the norovirus shell (S) domain that naturally builds the interior shells of norovirus capsids, Applicant has developed methods for the production of 60-valent, icosahedral S60 particles through the simple E. coli system. This can been achieved by several modifications to the S domain, for example an R69A mutation to destruct an exposed proteinase cleavage site and triple cysteine mutations (V57C/Q58C/S136C) to establish inter-S domain disulfide bonds for enhanced inter-S domain interactions. The polyvalent S60 particle with 60 exposed S domain C-termini offers an ideal platform for antigen presentation, leading to enhanced immunogenicity to the displayed antigen for vaccine development. This was proven by constructing a chimeric S60 particles displaying 60 rotavirus (RV) VP8 proteins, the major RV neutralizing antigens. These S60-VP8 particles are easily produced and elicited high IgG response in mice toward the displayed VP8 antigens. The mouse antisera after immunization with the S60-VP8 particles exhibited high blockades against RV VP8 binding to its glycan ligands and high neutralizing activities against RV infection in culture cells. The three-dimensional structures of the S60 and S60-VP8 particles were studied. Finally, the S60 particle can also display other antigens, supporting the notion that the S60 particle is a multifunctional vaccine platform.
Disclosed herein are methods and compositions that can be used to form a polyvalent vaccine composition, in particular, using a modified norovirus S particle.
In one aspect, a polyvalent icosahedral composition for antigen presentation is disclosed. The composition may comprise an S particle, wherein the S particle may comprise a recombinant fusion protein comprising a norovirus (NoV) S domain protein: a linker protein domain operatively connected to the norovirus S domain protein; and an antigen protein domain operatively connected to the linker.
The composition typically has an icosahedral symmetry structure. In one aspect, the composition comprises 60 sites for antigen presentation.
In one aspect, the norovirus S domain protein is that of a calicivirus. The calicivirus may be characterized by having 180 copies of a single capsid protein.
In one aspect, the norovirus S domain protein may comprise a mutation in a proteinase cleavage site of the NoV S domain protein, wherein the mutation renders the site resistant to trypsin cleavage. One or more mutations may be made to the site, provided the mutation effectively destroys the trypsin cleavage site. Modifications to the site that achieve such effect will be readily understood by one of ordinary skill in the art. In one aspect, the mutation may be at position 69 or position 70. In one aspect, the mutation may occur at position R69. In certain aspects, the mutation may be a change to any amino acid other than K (lysine), which is sufficient to destroy the proteinase cleavage site. In certain aspects, the mutation is R69A. In other aspects, the mutation may occur at position N70, for example, the mutation may be any amino acid other than P (proline) sufficient to destruct the proteinase cleavage site.
In one aspect, the norovirus S domain protein may comprise a mutation sufficient to provide a non-native disulfide bond binding site. The norovirus S domain protein may comprise a mutation of at least two amino acids (that are sterically close to each other) to cysteine residues sufficient to provide at least one non-native disulfide bond binding site, or, in other aspects, at least two non-native disulfide bond binding sites, or at least three non-native disulfide bond binding sites between neighboring S domain proteins of the polyvalent icosahedral S particle. In certain aspects, the mutation may be selected from V57C, Q58C, S136C, M140C, or a combination thereof.
In one aspect, the linker may comprise an amino acid sequence of a length sufficient to provide space and certain flexibility between the S domain protein particle and the displayed antigens. The linker is typically a short peptide of one to ten amino acid units, or three to six amino acids, that connect the C-terminus of the S domain to the displayed antigens. The linker provides space and certain flexibility between the S60 particle and the displayed antigens, which helps the independent folding of the S domain and the displayed antigens. A longer linker may be used as necessary. The amino acid length of the linker should be sufficient to allow flexibility of the protein domains to form the claimed compositions.
The disclosed compositions are ideally suited for presentation of an antigen. Suitable antigens may be readily determined by one of ordinary skill in the art. Exemplary antigens are disclosed herein. In certain aspects, the antigen protein domain may be selected by size, in addition to immunogenicity, and may encode for an antigen having a size of from 8 amino acids up to about 300 amino acids, or from 8 amino acids up to about 400 amino acids, or from 8 amino acids up to about 500 amino acids. As will be readily appreciated by one of ordinary skill in the art, the size of an antigen may vary greatly, and the instant compositions may be used for presentation of a variety of different antigens to illicit an immune response.
In one aspect, the polyvalent icosahedral composition may comprise an antigen protein domain that is a rotavirus (RV) antigen. In one aspect, the antigen protein domain may comprise an RV spike protein antigen (VP8 antigen). In further aspects, the antigen may comprise a TSR antigen of circumsporozoite protein (CSP) of malaria parasite Plasmodium falciparum, a receptor-binding domain of the HA1 protein and an M2e epitope of influenza A virus, a P domain antigen of hepatitis E, a surface spike protein of the astrovirus, and combinations thereof. Again, such antigens are merely exemplary and the recitation of such is not intended to limit the scope of the claims. Exemplary sequences include those of SEQ ID NO 34 and SEQ ID NO 35: human Rotavirus VP8 antigen, SEQ ID NO 42 and SEQ ID NO 43: P domain antigen of hepatitis E virus (HEV), SEQ ID NO 44 and SEQ ID NO 45: Surface Spike protein antigens of an avian AstV (see, e.g., (GenBank AC #: NP987088, residue 423-630), SEQ ID NO 46 and SEQ ID NO 47: HA1 antigen (H7) of influenza A virus, SEQ ID NO 48 and SEQ ID NO 49: TSR antigen of the circumsporozoite protein of Plasmodium falciparum, and SEQ ID NO 50 and SEQ ID NO 51: M2E epitope of influenza A viruses. It will be understood that antigen sequences used to generate the antigen peptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference nucleic acid sequence, provided that the resulting antigen elicits at least a partial immune response in an individual administered the composition having the antigen.
The recombinant fusion protein is a subunit of the disclosed vaccine compositions. Further disclosed herein are recombinant fusion proteins that may form the basis of the polyvalent icosahedral compositions. The fusion protein may comprise a norovirus (NoV) S domain protein having a mutation to the trypsin site as described above an added cysteine site as described above: a linker protein domain operatively connected to said norovirus S domain protein having the aforementioned mutations; and an antigen protein domain operatively connected to the linker. The features of each portion of the fusion proteins are described above.
In addition to the S particle described above, the disclosed compositions may further comprise one or more pharmaceutical-acceptable carriers, which may include any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The disclosed S particles may be provided in physiological saline. Optionally, a protectant may be included, for example, an anti-microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. The compositions may further include a stabilizing agent, such as for example saccharides, trehalose, mannitol, saccharose and the like, to increase and/or maintain product shelf-life. Those of skill in the art will understand that the composition herein may incorporate known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as e.g. saline or corresponding plasma protein solutions are readily available. In addition, the immunogenic and vaccine compositions of the present invention can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others. Suitable adjuvants will be appreciated by one of ordinary skill in the art.
In one aspect, disclosed is a container comprising at least one dose of the immunogenic compositions disclosed herein. The container may comprise 1 to 250 doses of the immunogenic composition, or in other aspects, 1, 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition. In one aspect, each of the containers may comprise more than one dose of the immunogenic composition and may further comprise an anti-microbiological active agent. Those agents may include, for example, antibiotics such as Gentamicin and Merthiolate and the like.
A further aspect relates to a kit. The kit may comprise any of the containers described above and an instruction manual, including the information for the delivery of the immunogenic composition disclosed above. For example, instructions related to intramuscular application of at least one dose may be provided for lessening the severity of clinical symptoms associated with an infection of an antigen as disclosed here. The kits and/or compositions may further include an immune stimulant such as keyhole limpet hemocyanin (KLH), or incomplete Freund's adjuvant (KLH/ICFA). Any other immune stimulant known to a person skilled in the art may also be used.
In one aspect, a method of making the disclosed polyvalent icosahedral structures are disclosed. The method may comprise the steps of a) making a first region comprising a modified NoV S domain protein, wherein said modification comprises a mutation sufficient to destruct an exposed protease cleavage site (wherein the mutation prevents protein degradation), preferably an R69A mutation, and introducing one or more mutations in said norovirus (NoV) S domain protein sufficient to create an inter-S domain protein disulfide bonds, for example, a mutation selected from V57C, Q58C, S136C and M140C, and combinations thereof, and b) recombinantly expressing the first region having a modified NoV S domain protein with a linker and an antigen. In certain aspects, the composition may be effectively produced in E. coli.
In one aspect, a method of eliciting an immune response in an individual in need thereof is disclosed. In this aspect, the method may include the step of administering a vaccine composition as disclosed above to an individual in need thereof. It will be readily appreciated that the disclosed compositions may be administered to an individual according to any method known in the art, and that optimal administration (including route and amounts) will not require undue experimentation. The vaccine compositions may be administered prophylactically to an individual suspected of having a future exposure to the antigen incorporated into the vaccine composition. In certain aspects, provided is a method of providing an immune response that protects an individual receiving the composition from infection, or reduces or lessens the severity of the clinical symptoms associated from an infection. The infection may include, for example, malaria, influenza A, hepatitis E, and an astroviral infection. Dosage regimen may be a single dose schedule or a multiple dose schedule (e.g., including booster doses) with a unit dosage form of the composition administered at different times. The term “unit dosage form.” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the antigenic compositions disclosed herein in an amount sufficient to produce the desired effect, which compositions are provided in association with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable diluent, carrier or vehicle). The vaccine may be administered in conjunction with other immunoregulatory agents.
The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Generations of novel biomaterials through bioengineering have been a fast-growing field in modern medicine. Typical examples include various polyvalent protein nanoparticles and complexes that have been constructed by taking advantage of the self-formation features of viral capsid proteins [1-3]. Viral capsid proteins are responsible for many basic functions necessary for viral life cycles, particular viral attachment and entry, and thus are able to elicit neutralizing antibodies against viral infection after immunization to humans and animals. This supports the notion that viral capsid proteins are excellent vaccine targets against corresponding viral pathogens. In fact, various capsid protein particles and complexes have been developed and used as non-replicating subunit vaccines to combat against different infectious diseases [1-3] that claim millions of lives each year. Unlike the traditional live-attenuated and inactivated virus vaccines that need cultivation of infectious virions and thus are associated with certain safety concerns, the non-replicating subunit vaccines that derived from bioengineered viral capsid proteins do not involve in an infectious agent, and therefore are safer with lower manufacturing costs than the traditional vaccines. Thus, non-replicating subunit vaccines represent a new generation of innovative vaccine strategy.
Plasmid constructs. 1) Expression construct of glutathione-s-transferase (GST)-tagged S domain protein. The S domain with the hinge-encoding sequences of a GII.4 November strain VA387 (GenBank AC #: AY038600.3; residue 1 to 221) were inserted into the multiple cloning sites of the pGEX-4T-1 vector (GST Gene Fusion System, GE Healthcare Life Sciences) via the BamH1/Sal I sites. The resulting S domain protein had an N-terminal GST with a thrombin cleavage site in between. 2) Plasmid construct for the His×6-fused SR69A domain expression. The same NoV S domain-hinge-encoding sequences with an R69A mutation were inserted into the multiple cloning sites of the pET-24b vector (Novagen) via the BamH1/Not I sites. The resulting S domain protein had a C-terminally fused His×6 peptide. 3) DNA construct for SR69A-VP8 (SEQ ID NO: 29) chimeric protein expression. A DNA fragment containing RV VP8-encoding sequences of a P[8] human RV strain BM14113, equivalent to the amino acid sequences from 64 to 231 of the VP8 of WA strain (GenBank AC #: VPXRWA), was fused to the C-terminal end of the SR69A domain-hinge with a linker (four histidines) in between. RV strain BM14113 was isolated directly from a RV positive stool sample [45]. A His×6-peptide was added to the C-terminus of the VP8 antigen for purification purpose. 4) Expression constructs of the SR69A/V57CM140C-VP8 (SEQ ID NO: 31), the SR69A/V57C/Q58C/S136C-VP8 (SEQ ID NO: 33), and SR69A/V57C/Q58C/S136C/M140C-VP8 (SEQ ID NO: 37) chimeric proteins. This DNA construct was made by introduction of other two (V57C/M140C), three (V57C/Q58C/S136C), or four (V57C/Q58C/S136C/M140C) mutations to the expression construct of the SR69A-VP8 (SEQ ID NO: 29) chimeric proteins through site-directed mutagenesis. 5). Plasmid construct for SR69A/V57C/Q58C/S136C-mVP8 (SEQ ID NO: 41) chimeric protein expression. This construct contained the DNA sequences like the SR69A/V57C/Q58C/S136C-VP8 (SEQ ID NO: 33) construct, but the VP8-encoding sequences were replaced with those encoding the VP8 of the murine RV EDIM (epizootic diarrhea of infant mice) strain [50]. In addition, DNA constructed for other SR69A/V57C/Q58C/S136C-based chimeric particles displaying antigens of various pathogens, including the surface TSR antigen of the circumsporozoite protein (CSP) (GenBank AC #:CAB38998, residues 309-375) of Plasmodium falciparum parasite 3D7 strain [51], the M2e epitope of influenza A virus [52, 53], and the P domain antigens of hepatitis E viruses [54-56], were constructed using the construct of SR69A/V57C/Q58C/S136C-VP8 (SEQ ID NO: 33) chimeric proteins as the starting construct, in which the RV VP8-encoding sequences are replaced with those encoding the corresponding antigens.
Production and purification of recombinant proteins. The recombinant GST- and Hstx6-fused proteins were expressed in E. coli (BL21, DE3) as described previously [11, 47, 53, 56]. The resulting recombinant proteins were purified using Sepharose 4 Fast Flow purification resin (GE Healthcare Life Sciences) for GST tagged and the TALON CellThru Resin (ClonTech) for the His×6-peptide fused proteins according to the instructions of the manufacturers. The GST can be removed from the target proteins by thrombin (GE Healthcare Life Sciences) cleavage, while the GST-fusion proteins still bound to the purification resin.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein quantitation. Purified proteins were analyzed by SDS-PAGE using 10% separating gels. Proteins were quantitated by SDS-PAGE using serially diluted bovine serum albumin (BSA, Bio-Rad) as standards on same gels [46].
Gel filtration chromatography. This was performed to analyze the size distributions of proteins and protein complexes as described previously [11, 53, 57] through an AKTA Fast Performance Liquid Chromatography System (AKTA Pure 25 L, GE Healthcare Life Sciences) using a size exclusion column (Superdex 200, 10/300 GL, GE Healthcare Life Sciences). The column was calibrated by gel-filtration calibration kits (GE Healthcare Life Sciences) and purified NoV P particles (˜830 kDa) [57], small P particles (˜420 kDa) [20], and P dimers (˜69 kDa) as described previously [53, 55]. The proteins of the elution peaks were analyzed by SDS-PAGE.
Cesium chloride (CsCl) density gradient ultracentrifugation. 0.5 mL of resin-purified S60-VP8 particles were mixed with 11 mL CsCl solution with a density of 1.300 g/mL and then packed in a 12-ml centrifuge tube. After centrifugation at 288,000 g for 45 h in the Optima L-90K ultracentrifuge (Beckman Coulter) using a SW41Ti rotor, the gradient was fractionated by bottom puncture into 23 fractions with 0.5 mL each. CsCl densities of fractions are determined based on the refractive index. The S60-VP8 particles in factions were analyzed by ELISA, in which individual fractions were diluted 20 folds in PBS and coated on microtiter plates. The coated proteins were detected by NoV VLP- and RV VP8-specific antibodies.
Electron microscopy. Protein samples were prepared for electron microscopy (EM) inspection for particle formation using 1% ammonium molybdate as the staining solution [22]. Specimens were observed under an EM10 C2 microscope (Zeiss, Germany) at 80 kV at a magnification of 10,000× to 40,000×.
Electrospray ionization mass spectrometry (ESI-MS). All ESI-MS measurements were carried out in positive ion mode using a Synapt G2S quadrupole-ion mobility separation-time of flight (Q-IMS-TOF) mass spectrometer (Waters, Manchester, UK) equipped with a nanoflow ESI (nanoESI) source. Each sample solution was prepared in 200 mM aqueous ammonium acetate buffer (pH 6.8, 25° C.) and loaded into a nanoESI tip, which was produced from borosilicate capillaries (1.0 mm o.d., 0.68 mm i.d.) pulled to ˜5 μm using a P-1000 micropipette puller (Sutter Instruments, Novato, CA). To perform ESI, a platinum wire was inserted into the nanoESI tip and a voltage of 1.10 KV was applied. A source temperature of 60° C. was used. The Cone, Trap and Transfer voltages were 50 V, 5 V and 2 V, respectively, and the Trap gas flow rate was 6.0 mL min-1: all other parameters were set at their default values. Data acquisition and processing were performed using Waters MassLynx software (version 4.1).
N-terminal amino acid sequencing. The SDS-PAGE gel slice containing the cleaved S domain protein was cut out and N-terminal amino acid sequencing was conducted by 494 Procise Protein Sequencer/140C Analyzer from Applied Biosystem, Inc. at the Protein Facility of Iowa State University (http://www.protein.iastate.edu/).
Immunization of mice. BALB/c mice (Harlan-Sprague-Dawley, Indianapolis, IN) at 3-4 weeks of age were divided into four groups (N=6) for immunizations with following immunogens (10 μg/mouse): 1) S60-VP8 chimeric particles: 2) free VP8 protein: S60 particle without VP8 antigen; and 4) diluent (phosphate buffered saline, PBS, pH7.4) in the same volume. Immunization was performed intramuscularly three times with Inject Alum adjuvant (Thermo Scientific, 50 μl/mouse) at two-week intervals. Bloods were collected two weeks after the third immunization and sera were prepared from blood samples via a standard protocol.
Enzyme immunoassay (EIA). EIA was performed to measure the antibody titers of mouse sera after immunization with different immunogens, as described previously [46]. Gel-filtration-purified free VP8 antigen at 1 μg/mL was coated on 96-well microtiter plates and incubated with serially diluted mouse sera [47]. Bound antibodies were detected by goat-anti-mouse secondary antibody-HRP conjugates (MP Biomedicals, Inc). Antibody titers were defined by the end-point dilutions with a cutoff signal intensity of OD450=0.1.
HBGA binding assay. Synthetic oligosaccharide- and saliva-based HBGA binding assay's were performed to measure the binding function of the S60 particle displayed VP8 to their HBGA ligands [45]. Briefly, synthetic oligosaccharides (2 μg/mL) representing the H1, Leb, and Ley antigens, respectively, or boiled saliva samples (1:1000 diluted) that are H1 and/or Leb positive or H1 and Leb negative were coated on 96-well microtiter plates and incubated with various S60-VP8 particles or S60 particles without RV VP8 at indicated concentrations. The bound proteins were measured by guinea pig anti-VP8 antiserum (for S60-VP8 particles) or guinea pig hyperimmune serum against GII.4 November VLPs (for S60 particles), followed by an incubation of HRP-conjugated goat anti-guinea pig IgG (ICN Pharmaceuticals).
Serum blocking titers against RV VP8-HBGA ligand attachment. This was performed as a surrogate neutralization assay as described previously [47]. Boiled and diluted (1:1000) human saliva samples with positive H1 and Lewis b (Leb) antigens, the ligands of P[8] RV [45, 58], were coated on microtiter plates. The P particle-displayed RV VP8 (PP-VP8) at 625 ng/ml was pre-incubated with the post-immune sera after immunization with various immunogens (S60-VP8 particles, free VP8 antigens, S60 particles, and PBS) at different dilutions before the PP-VP8 was added to the coated saliva samples. The 50% blocking titer (BT50) was defined as the lowest serum dilution causing at least 50% reduction against the binding of PP-VP8 particles to HBGAs/saliva samples compared with the unblocked positive control.
RV neutralization assay: This was performed as described previously [53]. Briefly, MA104 cells were cultivated in 6-well plates and tissue culture-adapted RV Wa strain (G1P[8]) at a titer of ˜50 PFU/well was used as the inoculum. Trypsin-treated Wa RVs were incubated with mouse sera after immunization with indicated immunogens (see above) for one hour and then was added to the cells. The plates were overlaid with media including trypsin (Invitrogen) and 0.8% agarose. After a four-day incubation, the plaques were stained and counted. The neutralization (%) of the sera was calculated by the reduction in plaque numbers in the wells treated with antisera relative to the number in untreated control wells.
Structural modeling of the S60 particle. The structures of the S60 particles with or without His×6 peptide and the S60-VP8 chimeric particles were modeled using the crystal structure (PDB #: 4PB6) of the 60-valent feline calicivirus (FCV) VLPs as template using software PyMOL Molecular Graphics System, version 1.8.2.0 (Schroinger, LLC). All crystal structure-based images were made by this software.
Structural reconstruction of the S60-VP8 chimeric particles by cryoEM. This was performed using a similar cryo-EM approach described in Applicant's previous studies [20, 21, 46]. Briefly, aliquots (3 to 4 μL) of gel-filtration-purified S60-VP8 chimeric particles were flash frozen onto Quantifoil grids that were then loaded into the microscope. Low-electron (e)-dose images (˜20 e/A2) were recorded on films using a CM200 cryomicroscope at a nominal magnification of ×50,000 and in the defocus range of 2.0 to 4.0 μm. The micrographs were selected and digitized by using a Nikon Super CoolScan 9000ED scanner at step size of 6.35 μm/pixel. The scanned images were binned, resulting in the final sampling of the images at 2.49 Å/pixel. The images of the S60-VP8 chimeric particles were selected using EMAN's boxer program. The selected images were manually filtered to exclude false positives. The EMAN's ctfit program was used to manually determine the contrast-transfer-function (CTF) parameters associated with the set of particle images originating from the same micrograph. Initial models of the particles were created using EMAN's startoct program. Then, the EMAN's refining program was used to iteratively determine the center and orientation of the raw particles and reconstruct the 3-D maps from the 2-D images by the EMAN make3d program until convergence. Icosahedron symmetry was imposed during reconstruction of the S60-VP8 chimeric particles. Analysis of cryo-EM models, including fitting of the S60 particle model (see aboe) and the crystal structure of P[8] RV VP8 (2DWR) were performed using UCSF Chimera software (version 1.12: http://www.rbvi.ucsf.edu/chimera).
Statistical analysis. Statistical differences among data sets were calculated by software GraphPad Prism 6 (GraphPad Software, Inc) using an unpaired, non-parametric t test. P-values were set at 0.05 (P<0.05) for significant difference, 0.01 (P<0.01) for highly significant difference, and 0.001 (P<0.001) for extremely significant difference.
Ethics statement. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (23a) of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Cincinnati Children's Hospital Research Foundation (Animal Welfare Assurance no. A3108-01).
Low particle formation efficiency of native NoV S domains. Applicant's study started with production of the native S domain with the hinge of a GII.4 November (VA387) in E. coli using the expression vector pGEX-4T-1, resulting in GST-S domain fusion proteins with a molecular weight (MW) of ˜51 kDa (
Applicant also observed a minor protein band with lower MW that co-occurred with the GST-S fusion proteins (
Identification of the exposed protease cleavage site in the S protein. The above findings prompted us to identify the protease cleaved site. This was achieved by N-terminally sequencing of the two cleaved S protein bands at ˜16 kDa (
Destruction of the protease site for high S particle formation efficiency. Based on the above data, Applicant introduced an R69A mutation to destroy the proteinase cleavage site, resulting in the SR69A (SEQ ID NO: 27) protein. In addition, Applicant used a C-terminally linked His×6 peptide to replace the GST tag to avoid the thrombin cleavage step for a simplified purification procedure (
The SR69A (SEQ ID NO: 27) protein (˜25 kDa) was produced well in the E. coli system and could be purified by the His×6-binding TALON CellThru Resin at extremely high yield (>40 mg/liter bacterial culture) and high stability (
Self-assembly of the SR68A (SEQ ID NO: 27) protein into 60-valent S60 particles. Applicant then performed ESI-MS analysis to determine the complexity of the SR69A (SEQ ID NO: 27) proteins, revealing three protein forms: 1) S monomers at 25.047 kDa, 2) S dimers at 50.095 kDa, and 3) S particles at ˜1.47 mDa (
Structural modeling of the S60 particle. While detailed structure of a 60-valent NoV capsid or its interior shell remains unknown, the crystal structure of the 60-valent feline calicivirus (FCV) VLP has been reported [59], providing a way to model the S60 particles to understand their structure features. The structure model of the S60 particle was built by using the crystal structure of the 60-valent FCV VLP (PDB #: 4PB6) as a template (
Applicant also modeled the S60 particle with C-terminal linkers (GGGG) (SEQ ID NO: 53) and His×6 peptides to mimic antigen presentation by the S60 particle (
Production and characterization of S60-VP8 chimeric particles. To prove the feasibility of the S60 particle as a platform for antigen presentation for enhanced immunogenicity, Applicant produced S60-VP8 chimeric particles that display RV VP8 proteins, the major RV neutralizing antigens. This was achieved by fusion of the RV VP8 protein to the C-terminus of the SR69A (SEQ ID NO: 27) protein via a linker (
EM observation of the proteins from peak 1 revealed many SR69A-VP8 particles (referred as S60-VP8 particles) in unified size with recognizable protrusions due to the surface displayed VP8 proteins, leading to rough surfaces (
Further stabilization of the S60-VP8 particles. The relatively low particle formation efficiency by the SR69A-VP8 proteins indicated room for improvement by increasing inter-molecular interaction between neighbor S domains in the S particles. Inspection of a GII.4 shell structure (W.J., unpublished data) indicated that V57 and Q58 of an S domain are sterically close to M140 and S136 of the neighboring S domain, respectively, with distances of 5.7 to 5.9 Å (
When V57 and M140 of the SR69A-VP8 (SEQ ID NO: 29) protein were mutated into cysteines (
When V57, Q58, and S136 of the SR69A-VP8 (SEQ ID NO: 29) protein were mutated into cysteines (
Structures of the S60-VP8 particles. Applicant constructed the three-dimensional (3-D) structure of the S60-VP8 particle by cryo-EM technology (see materials and methods) to a resolution of 14 Å, exhibit a T=1 symmetry containing 60 S-VP8 proteins (
When the crystal structure of the 60-valent FCV shell (PDB #: 4PB6) was fitted into the S60-particle portion of the S60-VP8 particle cryoEM density map, both structures fitted very well each other (
Applicant then fitted 60 copies of the VP8 crystal structure (PDB code: 2DWR) of the P[8] RV Wa strain into the protruding regions of the S60-VP8 particle cryoEM density maps (
The S60 particle displayed VP8s retain ligand-binding function. Applicant's previous study showed that VP8 of P[8] RV bound H1 antigens, but not Ley antigen [45]. Saliva-based binding assay indicated that S60-VP8 particles bound the H1 and/or Leb antigen-positive saliva samples, but not those that were negative for H1 and Leb antigens. These data indicated that the S60 particle-displayed VP8 antigens are in correct folding with ligand-binding function, validating the S60-VP8 particle as a RV vaccine candidate.
Improved immunogenicity toward the S60 particle-displayed VP8 antigens. The S60-VP8 particles were immunized to mice (N=6) and measured the VP8-specific immune responses using the free VP8 antigen as control for comparison. After three immunizations, the VP8-specific IgG response after immunization with the S60-VP8 particles was 11.6 folds higher than that induced by the free VP8 (P=0.0004) (
The S60-VP8 particle-elicited antisera enhanced blockade against VP8-ligand binding. Binding of VP8s to RV host ligands or receptors is a key step in RV infection [43]. Accordingly, an in vitro blocking assay against the binding of RV VP8 proteins to HBGAs has been developed as a surrogate RV neutralization assay [47]. Applicant performed such blocking assays using the previously developed P-VP8 particles and Leb-positive saliva samples as the RV ligands [45]. Applicant found that the mouse antisera after immunization with the S60-VP8 particles exhibited 22.8 folds higher 50% blocking titer (BT50) than that of the antisera after immunization with the free VP8 antigens (P=0.0003) (
The S60-VP8 particle-elicited antisera enhanced neutralization against RV infection. Applicant also performed conventional cell culture-based neutralization assays to determine the neutralizing activity of the S60-VP8 particle-elicited antisera against infection of the cell-culture adapted (P[8]) RV Wa strain. In consistence with their BT50s (see above), the mouse antisera after immunization with the S60-VP8 particles exhibited significantly higher neutralizing activities at three different serum dilutions (1:75, 1:150, and 1:300) than those of the antisera after immunization with the free VP8 antigens (P=0.0003, P=0.0001, and P=0.0016, respectively) (
The S60 particle as a multifunction vaccine platform. In addition to the RV VP8 antigen, Applicant have been able to fuse several other epitopes and antigens to the S60 particle through the same exposed S domain C-terminus via a linker, including the M2e epitope of influenza A virus, the TSR antigen of the circumsporozoite surface protein (CSP) of malaria parasite Plasmodium falciparum, and the P domain of hepatitis E virus (Table 1). Thus, the artificially developed S60 particle serves as a multifunction platform for novel vaccine development.
1M2e epitope is the ectodomain of Matrix-2 (M2) protein forming the proton-selective ion channel of an influenza A virus.
2TSR/CSP antigen is the C-terminal antigen of the major surface protein of a circumsporozoite (CSP) that play a key role in host cell invasion of a malaria parasite plasmodium falciparum.
3Full RV VP8 is the full-length VP8 domain of the spike protein of a human P[8] rotavirus. 3Murine RV VP8 is the core portion of the spike protein of a murine rotavirus.
4HEV P domain is the protruding domain of a hepatitis E virus capsid.
In this study Applicant have developed a new technology to produce unified 60-valent NoV S60 particles in a high efficiency via the simple bacterial expression system. This was achieved by taking advantage of the homotypic interactions of NoV VP1 S domain that naturally builds the interior shells of NoV capsids, as well as several modifications to stabilize the S domain proteins and enhance the inter-S domain interactions. Specifically, Applicant introduced an R69A mutation to destruct the exposed protease cleavage site on the native shells that otherwise leads to easy degradation of the S proteins. In addition, Applicant introduced triple (V57C/Q58C/S136′C) or quadruple (V57C/Q58C/S136′C/M140′° C.) cysteine mutations to two pairs of sterically close residues (V57/M140′ and Q58/S136′,
The self-assembled, polyvalent S60 particle with 60 flexibly exposed S domain C-termini is an ideal platform of antigen presentation for improved immunogenicity toward the displayed antigens for vaccine development. This idea has been largely proven in this study by constructing a chimeric S60 particles displaying 60 RV VP8 proteins, the major RV neutralizing antigens. The S60-VP8 particles can be easily produced with high stability. They elicited significantly higher IgG response in mice toward the displayed VP8 antigen than that induced by the free VP8 proteins. The mouse antisera after vaccination with the S60-VP8 particles exhibited significantly stronger blockade against RV VP8 binding to its glycan ligands and significantly higher neutralizing activities against RV infection and replication in culture cells than those of sera after immunization with the free VP8 antigens. While protective efficacy of the S60-VP8 particle vaccine is being determined using a murine RV challenge model in Applicant's lab, the presented data in this report strongly supported the notion that the S60-VP8 particle is a promising vaccine candidate against RV infection and thus the S60 particle is an excellent platform for antigen display for novel vaccine development.
Native NoV capsids are made by 180 VP Is that are the single major structural protein of NoVs. In vitro expression of NoV VP1 via a eukaryotic system often resulted in a mixture of 180- and 60-valent VLPs and the two VLP formats were exchangeable via artificial denature and renature treatments [60]. Although it has not yet been thoroughly studied, previous expression of truncated S domains via the baculovirus/insect cell system appeared to self-assemble 180-valent S particles [11, 24]. However, unified 60-valent NoV VLPs or S particles via an expression system has never been produced previously. Therefore, Applicant's production technology of unified NoV S60 particles via the simple E. coli system represents a bioengineering advancement. The self-formation of the unified S60 particles may result from the combined impacts of the heavily modified S domain and the unique folding environment of the prokaryotic E. coli expression system. Homogenous complexity and size of a vaccine candidate is an important consideration for quality control, because variations in vaccine complexity and size will lead to variations in immunization outcomes of the vaccine.
Artificially introduced inter-molecular disulfide bonds may be used as a general approach to stabilize a viral protein particle or complex. During Applicant's previous construction of NoV P particles, Applicant found that addition of a cysteine-containing peptide to the end of NoV P domain promoted and stabilized P particle formation via inter-P dimer disulfide bonds [20-23]. In this current study, the S60 particles self-assembled efficiently (
Among these mutations, the S60-VP8 particles with the triple cysteine mutations (V57C/Q58C/S136′C) exhibited the highest particle formation efficiency with >95% the S-VP8 proteins self-assembling into the S60-VP8 particles (
The S60- and S60-VP8 particles in this study were purified via a small His×6 peptide that was linked to the exposed C-terminus of the S domain or the S-VP8 protein. Applicant's data showed that the GST tag is not suitable for the S60- and S60-VP8 particle production, because it is large (220 residues), disturbing the S60 particle formation, and thus needs to be removed by an extra thrombin cleavage step, greatly complicating the purification procedure. In addition, a tag-free purification method was tested. Applicant found that the both S60 and S60-VP8 particles can be selectively precipitated by ammonium sulfate and resolved in PBS and other buffers (data not shown). Finally, Applicant discovered that the S60 and the S60-VP8 particles were eluted as a single peak through the gel-filtration size exclusion column and in anion exchange chromatography (data not shown). These data collectively indicated that the S60 and the S60-VP8 particles, and most likely other S60-antigen chimeric particles can be purified through a tag-free approach.
The 60 freely exposed C-termini are another feature facilitating the S60 particle to be a useful vaccine platform. Foreign antigens or epitopes can simply be fused to the end of the S domain via a flexible linker through recombinant DNA technology. This study showed clearly that the His×6 peptide and the RV VP8 antigen can be presented well by the S60 particle, as shown by the structural stability of the S60-His×6 and the S60-VP8 particles, as well as by their excellent binding abilities to the TALON CellThru Resin (His×6) and the H1 and Leb ligands (RV VP8). In addition, the fact that several other tested antigens or epitopes can be well presented by the S60 particles indicate the S60 particles as a multifunctional vaccine platform.
The modeling of the S60 particle, the S60-His×6 using the crystal structure of the 60 valent FCV VLP and the reconstruction of the 3-D structures of the S60-VP8 particle via cryoEM technology provide new insights into the structural basis of how the S60 particle displays the Hsix6 peptide and the RV VP8 antigen. Fitting the structure of the S60 particle model into the S60 particle region, as well as 60 copies of VP8 antigens into the protruding regions of the S60-VP8 particle cryoEM density map shed further light on the structural relationship between the S60 particle and their displayed antigens. These structural data will help design and understanding of future presentations of other foreign antigens by the S60 particle. Finally, these structural studies also confirmed the 60 valent T=1 icosahedral symmetry of the S60 particles and the S60-VP8 particles.
In summary, a self-assembled, polyvalent protein nanoparticle featured with easy production, high stability, and high immunogenicity was developed, serving as an ideal platform for antigen display. As a proof of concept, a chimeric S60 particle displaying 60 copies of RV neutralizing VP8 antigens has been constructed. Applicant's data indicated that the highly immunogenic S60-VP8 particle is a promising vaccine candidate against RV infection and that the S60 particle is a multifunctional platform to enhance immunogenicity of various antigens for novel vaccine development against different pathogens.
Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen. Nature 2012; 485:256-9.
All percentages and ratios are calculated by weight unless otherwise indicated.
All percentages and ratios are calculated based on the total composition unless otherwise indicated.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”
Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
This application is a continuation of U.S. application Ser. No. 16/489,095, filed Aug. 27, 2019, which is a National Stage Entry of International Application Serial No. PCT/US18/22552 filed Mar. 15, 2018, which claims priority to and benefit of U.S. Provisional Application Ser. No. 62/477,481, filed Mar. 28, 2017, the contents of each are incorporated in their entirety and for all purposes.
This invention was made with government support under AI092434, AI089634 and AI114831 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62477481 | Mar 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16489095 | Aug 2019 | US |
| Child | 18490287 | US |
