YOLK IMMUNOGLOBULIN (IgY) AGAINST COXSACKIEVIRUS A16 AND/OR ENTEROVIRUS A71 AND ITS PREPARATION AND USES THEREOF
20250250327 ยท 2025-08-07
Assignee
- National Health Research Institutes (Miaoli County, TW)
- National Defense Medical Center (Taipei City, TW)
Inventors
- Chia-Chyi LIU (Miaoli County, TW)
- Yu-Sheng SHEN (Miaoli County, TW)
- Chi-Hsun CHEN (Miaoli County, TW)
- Chia-Tsui YEH (Taipei City, TW)
- Cheng-Cheung Chen (Taipei City, TW)
- Xin-An Chen (Taipei City, TW)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to immunoglobulin yolk (IgY) against coxsackievirus A16 and/or enterovirus A71 and its preparation and uses thereof.
Claims
1. A method for preparing a yolk immunoglobulin (IgY) against coxsackievirus A6 (CVA16) and/or enterovirus A71 (EV-A71) comprising (i) immunizing an egg-laying avian with CVA16 particles selected from the group consisting of CVA16 empty (E) particles, CVA16 full (F) particles and a combination thereof to obtain an immunized egg product from the avian; and optionally (ii) extracting and purifying an anti-CVA16 IgY from the immunized egg product.
2. The method of claim 1, wherein the CVA16 particles are purified.
3. The method of claim 1, wherein the CVA16 particles are CVA 16 E particles.
4. The method of claim 1, wherein the CVA16 particles are CVA16 F particles.
5. The method of claim 1, wherein the CVA16 particles are CVA16 E particles and CVA16 F particles.
6. The method of claim 1, wherein the egg-laying avian is selected from the group consisting of chicken, turkey, goose, duck, pheasant, quail, pigeon and ostrich.
7. The method of claim 6, wherein the egg-laying avian is chicken.
8. The method of claim 1, wherein the CVA16 particles are formulated with an adjuvant.
9. The method of claim 1, wherein the immunizing of the egg-laying avian comprises administering the egg-laying avian with a first injection, followed by one or more booster injections, of the CVA16 particles.
10. The method of claim 1, wherein the CVA16 particles are CVA16 E particles and the anti-CVA16 IgY thus prepared neutralizes CVA16 and enterovirus A71 (EV-A71).
11. The method of claim 1, wherein the CVA16 particles are CVA16 F particles and the anti-CVA16 IgY thus prepared neutralizes CVA16 and does not neutralize enterovirus A71 (EV-A71).
12. An anti-CVA16 IgY prepared by the method of claim 1.
13. The anti-CVA16 IgY of claim 12, wherein the IgY specifically binds to (i) an epitope of capsid protein VP1 selected from the group consisting of VP1-1, VP1-2, VP1-5, VP1-6, VP1-14, VP-24, VP1-42, VP1-43, VP1-50 and any combination thereof, as shown in Table 2; (ii) an epitope of capsid protein VP2 selected from the group consisting of VP2-10, VP2-25, VP2-28, VP2-40 and any combination thereof, as shown in Table 2; (iii) an epitope of capsid protein VP3 selected from the group consisting of VP3-1, VP3-3, VP3-4, VP3-5, VP3-6, VP3-7, VP3-8, VP3-9, VP3-14, VP3-18, VP3-47 and any combination thereof, as shown in Table 2; and/or (iv) an epitope of capsid protein VP4 selected from the group consisting of VP4-10, VP4-11, VP4-12 and any combination thereof, as shown in Table 2.
14. The anti-CVA16 IgY of claim 13, wherein the IgY specifically binds to (i) an epitope of capsid protein VP1 selected from the group consisting of VP1-1, VP1-2, VP1-5, VP1-42, VP1-50 and any combination thereof, (ii) an epitope of capsid protein VP2 selected from the group consisting of VP2-25; and/or (iii) an epitope of capsid protein VP3 selected from the group consisting of VP3-1, VP3-4, VP3-5, VP3-6, VP3-7, VP3-8, VP3-9, VP3-14 and any combination thereof.
15. The anti-CVA16 IgY of claim 14, wherein the IgY does not bind to (i) an epitope of capsid protein VP1 selected from the group consisting of VP1-6, VP1-14, VP-24, VP1-43 and any combination thereof; (ii) an epitope of capsid protein VP2 selected from the group consisting of VP2-10, VP2-28, VP2-40 and any combination thereof, (iii) an epitope of capsid protein VP3 selected from the group consisting of VP3-3, VP3-18, VP3-47 and any combination thereof; and/or (iv) an epitope of capsid protein VP4 selected from the group consisting of VP4-10, VP4-11, VP4-12 and any combination thereof.
16. The anti-CVA16 IgY of claim 14-OF-15, wherein the anti-CVA16 IgY neutralizes CVA16 and enterovirus A71 (EV-A71).
17. The anti-CVA16 IgY of claim 12, wherein the IgY specifically binds to (i) an epitope of capsid protein VP1 selected from the group consisting of VP1-1, VP1-6, VP1-14, VP-24, VP1-42, VP1-43 and any combination thereof; (ii) an epitope of capsid protein VP2 selected from the group consisting of VP2-10, VP2-25, VP2-28, VP2-40 and any combination thereof; (iii) an epitope of capsid protein VP3 selected from the group consisting of VP3-3, VP3-4, VP3-18, VP3-47 and any combination thereof; and/or (iv) an epitope of capsid protein VP4 selected from the group consisting of VP4-10, VP4-11, VP4-12 and any combination thereof.
18. The anti-CVA16 IgY of claim 16, wherein the IgY does not bind to (i) an epitope of capsid protein VP1 selected from the group consisting of VP1-2, VP1-5, VP1-50 and any combination thereof, and/or (ii) an epitope of capsid protein VP3 selected from the group consisting of VP3-1, VP3-5, VP3-6, VP3-7, VP3-8, VP3-9, VP3-14 and any combination thereof.
19. The anti-CVA16 IgY of claim 17, wherein the anti-CVA16 IgY neutralizes CVA16 but does not neutralize enterovirus A71 (EV-A71).
20. A composition comprising the anti-CVA16 IgY of claim 12 and a carrier.
21. A method for treating or preventing CVA16 and/or EV-A71 infection, comprising administering to a subject in need thereof an effective amount of the anti-CVA16 IgY of claim 12 or a composition comprising the anti-CVA16 IgY and a carrier.
22. A method for diagnosing CVA16 and/or EV-A71 infection, comprising the steps of (i) providing a sample derived from a subject in need thereof; (ii) reacting the sample with the anti-CVA16 IgY of claim 12 or a composition comprising the anti-CVA16 IgY and a carrier; and (iii) detecting an antigen-antibody reaction between the anti-CVA16 IgY and CVA16 and/or EV-A71 present in the sample.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] The patent or 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.
[0055] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
[0056] In the drawings:
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DETAILED DESCRIPTION OF THE INVENTION
[0064] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
[0065] As used herein, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a component includes a plurality of such components and equivalents thereof known to those skilled in the art.
[0066] The term comprise or comprising is generally used in the sense of include/including which means permitting the presence of one or more features, ingredients or components. The term comprise or comprising encompasses the term consists or consisting of.
[0067] As used herein, the term antibody (interchangeably used in plural form, antibodies) means an immunoglobulin molecule having the ability to specifically bind to a particular target antigenic molecule. As used herein, the term antibody includes not only intact (i.e., full-length) antibody molecules but also antigen-binding fragments thereof retaining antigen binding ability (e.g., Fab, Fab, F(ab)2, and Fv). Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. The term antibody also includes chimeric antibodies, humanized antibodies, human antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including amino acid sequence variants of antibodies, glycosylation variants of antibodies, and covalently modified antibodies. Immunoglobulin Y (IgY, Y=yolk) refers to antibodies that are produced in the body of avian and can be extracted from egg yolk.
[0068] As used herein, the term viral particle can mean the fully or partially assembled capsid of a virus that may include an empty particle, a full particle or a sub-particle.
[0069] As used herein, the term full particle or F-particle is a virus particle containing viral genomic nucleic acids encapsulated within a protein shell (capsid). In particular, CVA 16 full particles include CVA16 genomic RNA encapsulated within the protein shell comprised of capsid proteins including viral capsid protein 1 (VP1), Viral capsid protein 2 (VP2), Viral capsid protein 3 (VP3) and Viral capsid protein 4 (VP4). More details are described in 1981 Putnak & Phillips:
[0070] As used herein, the term empty particle or empty capsid or E-particle is a virus particle comprising at least one capsid protein but lack the viral genome. In some embodiments, CVA 16 empty particles include VP0, VP1 and VP3 (without CVA16 genomic RNA). More details are described in 1981 Putnak & Phillips: Fig5 & Table 3.
[0071] As used herein, the term purifying, purification, isolate, isolating, or isolation as used herein, may refer to increasing the degree of purity of a target product (e.g. viral particles) from a sample comprising the target product and one or more impurities. Typically, the degree of purity of target viral particles is increased by removing (completely or partially) at least one impurity from the sample. In some embodiments, viral particles as described herein can be said to be purified if it is substantially free of cellular materials or viral components other than the target viral particles. In some cases, purified viral particles can include a preparation containing the viral particles having less than 50%, 40%, 30%, 20% or 10% (by weight) of cellular proteins and other viral components.
[0072] The term immunization as used herein refers to a process known in the art for inducing an immune response in an animal by introducing an antigenic agent or substance into the animal (e.g., by injection, by mucosal challenge, etc.), which preferably results in a specific immune response (e.g. generating specific antibodies) to the antigenic agent or substance. The antigenic agent or substance can be introduced to the animal, with or without the use of adjuvants.
[0073] As used herein, the term adjuvant refers to a substance added to an immunogenic composition comprising an antigenic agent that while not having any specific antigenic effect in itself, can stimulate the immune system and increase the immune response to the antigenic agent. Examples of adjuvants include, but are not limited to, alum-precipitate, Freund's complete adjuvant, Freund's incomplete adjuvant, monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant, water in oil emulsion containing Corynebacterium parvum and tRNA, and other substances that accomplish the task of increasing immune response.
[0074] As used herein, the term treatment refers to the application or administration of one or more active agents to a subject afflicted with a disorder, a symptom or condition of the disorder, or a progression of the disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom or condition of the disorder, the disabilities induced by the disorder, or the progression of the disorder.
[0075] As used herein, the term preventing refers to the preventive measures for a disease or the symptoms or conditions of a disease. The preventive measures include, but are not limited to applying or administering one or more active agents to a subject who has not yet been diagnosed as a patient suffering from the disease or the symptoms or conditions of the disease but may be susceptible or prone to the disease. The purpose of the preventive measures is to avoid, prevent, or postpone the occurrence of the disease or the symptoms or conditions of the disease.
[0076] As used herein, the terms subject, individual and patient refer to any mammalian subject for whom diagnosis, prognosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.
[0077] As used herein, the term diagnosis as used herein generally includes determination as to whether a subject is likely affected by a given disease, disorder or dysfunction. The skilled persons often make a diagnosis on the basis of one or more diagnostic indicators, i.e., a marker, the presence, absence, or amount of which is indicative of the presence or absence of the disease, disorder or dysfunction. It will be understood in the art that diagnosis does not mean determining the presence or absence of a particular disease with 100% accuracy, but rather an increased likelihood of the presence of certain disease in a subject.
[0078] The present invention provides a method for preparing an IgY against CVA16 by immunizing an egg-laying avian with CVA16 particles including CVA16 empty (E) particles and/or CVA16 full (F) particles. Specifically, the immunization is performed with purified CVA16 E particles and/or VA16 F particles. According to the present invention, the immunization is not performed with an infected cell lysate.
[0079] Viral particles can be produced by methods known in the art. In general, the production of viral particles requires suitable host cells, including for example, human-derived cell lines, such as human embryonic kidney cell (HEK) 293 cells or rhabdomyosarcoma (RD) cells, mammalian cell lines such as Vero, or insect-derived cell lines such as SF-9 in the case of baculovirus production systems; and suitable media including for example, Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and VP-SFM media. Particularly, suitable host cells are contacted with virus which results in viral infection in the cells; the infected cells are cultured for a period of time sufficient to allow for virus propagation and production of the vial particles; and then the viral particles thus produced are collected. More particularly, before infection, cells are cultured to reach a density of about 10.sup.5 to 10.sup.6 cells per mL and then the cells are infected with the virus at a MOI (multiplicity of infection) of about 10.sup.2 to 10.sup.5 and cultivated for about 3 to 10 days. The virus particles are then harvested from the culture supernatants, which are subjected to a subsequent procedure, such as concentration, purification and/or inactivation.
[0080] In some embodiments, the purification is conducted by liquid chromatography purification, sucrose gradient ultracentrifuge purification, or a combination thereof. Preferably, the purification is conducted by sucrose gradient ultracentrifuge purification. More preferably, 10 to 60% sucrose density gradient is used in the sucrose gradient ultracentrifuge purification.
[0081] In some embodiments, the purification is conducted to obtain a fraction of CVA16 empty (E) particles. In particular, a fraction of CVA16 empty (E) particles can be obtained from 19-27% sucrose gradient.
[0082] In some embodiments, the purification is conducted to obtain a fraction of CVA16 full (F) particles. In particular, a fraction of CVA16 full (F) particles can be obtained from 29-40% sucrose gradient.
[0083] In some embodiments, CVA16 E-particles as described herein has a molecular weight of about 5,000-7,000 kDa. In some particular embodiments, CVA16 E-particles as described herein has a molecular weight of about 5,460-6,060 kDa. In some embodiments, CVA16 E-particles as described herein include VP0 (35-38 kDa), VP1 (32-35 kDa), and VP3 (24-28 kDa).
[0084] In some embodiments, CVA16 F-particles as described herein has a molecular weight of about 7,500-8,500 kDa. In some particular embodiments, CVA16 F-particles as described herein has a molecular weight of about 7,836-8,436 kDa. In some embodiments, CVA16 F-particles as described herein include VP1 (32-35 kDa), VP2 (24-28 kDa), VP3 (24-28 kDa), VP4 (6-8 kDa) and viral RNA (RNA: 2,376 kDa).
[0085] In some embodiments, CVA16 E-particles and CVA16 F-particles as described herein can be obtained from CVA16 strains as known in the art. One certain example is CVA16 5079 (clinical isolate).
[0086] In some embodiments, CVA16 VP0 as described herein is from CVA16 5079 capsid protein VP0 having the amino acid sequence as set forth in SEQ ID NO: 28, or from other CVA16 strains having 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% to SEQ ID NO: 28.
[0087] In some embodiments, CVA16 VP1 as described herein is from CVA16 5079 capsid protein VP1 having the amino acid sequence as set forth in SEQ ID NO: 29, or from other CVA16 strains having 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% to SEQ ID NO: 29.
[0088] In some embodiments, CVA16 VP2 as described herein is from CVA16 5079 capsid protein VP2 having the amino acid sequence as set forth in SEQ ID NO: 30, or from other CVA16 strains having 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% to SEQ ID NO: 30.
[0089] In some embodiments, CVA16 VP3 as described herein is from CVA16 5079 capsid protein VP3 having the amino acid sequence as set forth in SEQ ID NO: 31, or from other CVA16 strains having 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% to SEQ ID NO: 31
[0090] In some embodiments, CVA16 VP4 as described herein is from CVA16 5079 capsid protein VP4 having the amino acid sequence as set forth in SEQ ID NO: 32, or from other CVA16 strains having 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% to SEQ ID NO: 32
[0091] In particular, the collected viral particles are inactivated, for example, by formalin treatment. In certain examples, the treatment with formaldehyde is at about 20-45 C. for 2 to 20 days.
[0092] To prepare an IgY as described in the present invention, CVA16 viral particles can be mixed with a proper adjuvant as known in this art (e.g. a complete Freund adjuvant) and then administered to a fowl via muscle injection, for example, for immunization. Further immunization of CVA16 viral particles with an incomplete Freund adjuvant, for example, is preferred to boost the immune response in the fowl. In general, one to three times of boost or more is suggested, and the period of time between each immunization is about 7 to 28 days. After the immunization, eggs laid by the fowl are collected and the yolk is isolated from the eggs. In some embodiments, the yolk is further processed as needed e.g. lyophilized for the purpose of easy storage. In some embodiments, the yolk is processed to perform immunoglobulin extraction from the yolk. Many prior art methods of immunoglobulin isolation are available involving use of precipitating agents, such as ethanol, polyethylene glycol, lyotropic salts such as copper sulphate, ammonium sulphate and ammonium phosphate, and caprylic acid. The presence and activity of specific antibodies in the yolk can be confirmed by any method such as an immunological assay known in art e.g. Enzyme-linked immunosorbent assay (ELISA), a method using agglutination reaction and/or a neutralizing test.
[0093] In some embodiments, the anti-CVA16 antibody thus prepared specifically recognizes various epitopes of CVA16 capsid proteins. See Table 2.
[0094] In some embodiments, the immunization is performed with CVA16 E particles and the anti-CVA16 IgY thus prepared neutralizes CVA16 and EV-A71.
[0095] In some embodiments, the immunization is performed with CVA16 F particles and the anti-CVA16 IgY thus prepared neutralizes CVA16 and fail to neutralize EV-A71.
[0096] According to the present invention, the anti-CVA16 antibody may be formulated with a carrier to form a composition for purpose of delivery and absorption.
[0097] As used herein, a carrier may be a pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable means that the carrier is compatible with an active ingredient in the composition, and preferably can stabilize said active ingredient and is safe to the receiving individual. Said carrier may be a diluent, excipient, matrix or vehicle to the active ingredient. Typically, a composition comprising an antibody as described herein as an active ingredient can be in a form of a solution such as an aqueous solution e.g. a saline solution or it can be provided in powder form. Appropriate excipients also include lactose, sucrose, dextrose, sorbose, mannose, starch, Arabic gum, calcium phosphate, alginates, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, sterilized water, syrup, and methylcellulose. The composition may further contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, for example, pH adjusting and buffering agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The form of the composition may be tablets, pills, powder, lozenges, packets, troches, elixers, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterilized injection fluid, and packaged powder. The composition of the present invention may be delivered via any physiologically acceptable route, such as oral, parenteral (such as intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. In certain embodiments, the composition of the present invention is administered as a liquid injectable formulation which can be provided as a ready-to-use dosage form or as a reconstitutable stable powder.
[0098] The anti-CVA16 antibody of the present invention exhibits neutralizing capacity against CAV16 and/or EV-A71 infection. Therefore, the present invention provides a method for treating or preventing CAV16 and/or EV-A71 infection by administering to a subject in need thereof an effective amount of an anti-CVA16 IgY as described herein.
[0099] The anti-CVA16 antibody of the present invention is also useful to diagnosis purposes. Therefore, the present invention provides a method for diagnosing CAV16 and/or EV-A71 infection. In particular, the method comprises (i) providing a sample derived from a subject in need thereof; (ii) reacting the sample with an anti-CVA16 IgY as described herein; and (iii) detecting an antigen-antibody reaction between the anti-CVA16 IgY and CAV16 and/or EV-A71 present in the sample.
[0100] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES
[0101] During the propagation of enterovirus in mammalian cells, many types of virus particles are produced. Among them, two major kinds of viral particles are usually harvested the empty (E)-particle (immature virion) and the full (F)-particle (mature virion), which present different immunogenicity in animal study. [Putnak and Phillips, 1981; Liu et al., 2011; Chong et al., 2012; Liu et al., 2016; Lien et al., 2023]. In this study, we immunized laying hens with purified E- and F-CVA16 viral particles, and then purified the CVA16-specific IgYs from egg yolks. We characterize these CVA16-specific IgYs by liner epitope mapping and in silico simulation. The results provide important information that differs from previous studies of mammalian models, and these CVA16-specific IgYs can be used as diagnostic tools and antiviral reagent for therapeutic application to detect and prevent CVA16 and Enterovirus A71 infection.
1. Material and Methods
1.1. Cells, Media and Viruses
[0102] Vero (CCL-81) cells were obtained from the American Type Culture Collection (ATCC, USA). Vero cells were grown in a VP-SFM medium (Gibco). The human rhabdomyosarcoma cell line (RD) was obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. RD cells were grown in a DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone). All cell lines were passaged twice weekly in T-flasks. The CVA16 5079 strain was obtained from National Cheng Kung University Hospital, Taiwan. The genomic sequence of CVA16 5079 was reported in GenBank: AF177911.1 (J Med Virol. 2001 October; 65 (2): 331-9; PLOS One. 2012 Nov. 30; 7 (11): e49973). The EV-A71 E59 strain was obtained from the Taiwan CDC. These virus stocks were prepared from the supernatants of infected RD cells at 3 days post-infection (DPI). The titers of virus were determined by the median tissue culture infectious dose (TCID.sub.50) assay.
1.2 CVA16 Viral Particle Preparation
[0103] Vero cells were grown using VP-SFM in 850 cm.sup.2 roller bottle for CVA16 propagation. The medium was replaced with 333 mL fresh medium and the cells were infected with CVA16 at the multiplicity of infection (MOI)=10.sup.4. After 8-day culture, CVA16 virus solution was harvested and concentrated. Two types of CVA16 viral particles (E-particle and F-particle) were purified using sucrose gradient ultracentrifugation according to previous study [Chong et al., 2012]. Specifically, the virus culture supernatant was harvested from the T-flask culture. The cell debris was removed by passage through a 0.65 m filter (Sartorius, Germany), and the supernatant was concentrated 20-fold with a 100K TFF capsule (Pall). The crude virus concentrate (50 mL) was loaded onto a 10-60% continuous sucrose gradient and centrifuged at 32,000 rpm for three hours using a zonal rotor in a Hitachi CP80 ultracentrifuge. The fractions (50 mL per fraction) at 10 to 60% sucrose were collected and individually dialyzed against three exchanges of 1 L PBS at pH 7.4 (Gibco/Life Technologies, Taipei, Taiwan), then stored at 4 C. The fractions were also subjected to SDS-PAGE and Western blot analyses. The fractions identified to contain virus were pooled and concentrated by diafiltration using an Amicon 100K tube (Millipore, Belerica, MA USA) and centrifuged at 3,000g, then stored at 4 C. The total protein concentration of the purified virus fractions was determined by a BCA protein assay. CVA16 empty (E) particles was identified at 19-27% sucrose gradient, and CVA16 full (F) particles was identified at 29-40% sucrose gradient. The resultant CVA16 viral particles were inactivated by 1/4000 (v/v) formalin at 37 C. for 3 days and stored at 4 C. EV-A71 bulk sample (S05), which composed of mix-particles, was used as control and produced by the PIC/S cGMP facility of the National Health Research Institutes, Taiwan.
1.3 Immunization of Laying Hens with CVA16 and EV-A71 Viral Particles
[0104] Each type of CVA16 viral particles were prepared in 0.5 mL of normal saline solution and then emulsified with 0.5 mL of Freund's complete adjuvant (Sigma-Aldrich). In CVA16 E-particle group, 2 chickens (A16-E1 and A16-E2) were immunized with 70 g/dose antigens; in CVA16 F-particle group, 2 chickens (A16-F1 and A16-F2) were immunized with 40 g/dose antigens; in EV-A71 bulk group, 2 chickens (EV-A71-B1 and EV-A71-B2) were immunized with 25 g/dose antigens. The resultant emulsified samples with complete Freund's adjuvant (V/V=0.5 mL+0.5 mL) were used for immunizing the laying hens via intramuscular injection into 5 different sites on the muscles of the thorax. Four weeks after the 1st immunization, the same antigen proteins formulated in incomplete Freund's adjuvant (V/V=0.5 mL+0.5 mL) were used for the 2nd immunization of these hens using the same procedures. Four weeks after the final injection, eggs were harvested and stored at 4 C.
1.4 the Extraction of Anti-CVA16 and Anti-EV-A71 IgYs
[0105] The extraction of IgYs was followed by previous report for extracting IgY from egg yolks [Yeh et al., 2022]. The method for extracting IgY from egg yolks is conducted as follows. Briefly, the yolk sample with the buffer solution was diluted with the acetate-based buffer solution with a pH ranging from 4.6 to 5.4. The mixture was then stirred for at least 30 min at 4 C., followed by centrifugation. After centrifugation of the mixture at 14,000g for 30 min, the supernatant was collected. Thereafter, the inorganic ammonium sulfate salt solution with a concentration ranging from 0.05 M to 0.15 M and a saturation degree from 30% to 60% was added into the supernatant to salt out the IgYs. After another stirring for 30 min, the mixture was centrifuged again at 14,000g for 30 min to precipitate the IgYs. The whole extraction process requires approximately 2 hours.
1.5 SDS-PAGE and Western Blotting Analysis
[0106] Western blotting analyses were performed using 10% Tris-Glycine SDS-PAGE. Samples were separated by SDS-PAGE and transferred onto a PVDF membrane (Invitrogen) using a Mini Trans-Blot Cell (Bio-Rad) according to the manufacturer's instructions. Primary antibodies such as anti-CV-A16 IgYs, anti-EV-A71 IgY, and previously described enterovirus-related commercial antibodies, were used to detect the enterovirus lysates. Secondary antibodies such as goat anti-rabbit IgG (AP132P, Millipore) conjugated with horseradish peroxidase (HRP), goat anti-mouse IgG HRP (GTX213111-01, GeneTex), and rabbit anti-chicken IgY HRP (A9046, Sigma Aldrich) were then applied to the membrane. Immobilon crescendo western HRP substrate WBLUR0500 (Millipore) was used for chemiluminescence development and the image was captured by the Amersham Imager 600 system (GE Healthcare).
1.6 Virus Neutralizing Test
[0107] The virus neutralization test (Nt) was performed as described previously [Liu et al., 2011]. Each sample was then serially diluted (2-fold) with culture medium. Two hundred L of virus solution with titer equal to 200 TCID.sub.50 were added to tubes containing 200 L of the diluted sera. After incubation at 4 C. for 18-24 hours, these samples (100 L/well) were added to 96-well plates containing RD cells. The cultures were incubated for 6 days at 37 C., and TCID.sub.50 were measured after quantifying the CPE in the infected RD cells. The Nt value is the geometric reciprocal of the serum dilution yielding a 50% reduction in the viral titer, was obtained using the Reed-Muench methods.
1.7 Specific Titer ELISA Analysis
[0108] Three chicken IgYs (Anti-CVA16-E IgY, Anti-CVA16-F IgY, and Anti-EV-A71 IgY) were diluted to recognize the formalin-inactivated viral particles (CVA16 E-particle, CVA16 F-particle, and EV-A71 bulk) by ELISA. Viral particles were coated on a 96-well ELISA plate (Corning) at 10 ng/well in 50 L coating buffer (1M NaHCO.sub.3, pH9.5) (Liu et al., 2016). After antigen coating, 250 L of 5% skim milk in PBS was added for blocking. One hundred L of testing sera and antibodies were added to testing wells and incubated for 2 hours at room temperature. The wells were washed four times with 250 L wash buffer (PBS+0.05% Tween20), and 100 L of an HRP-conjugated secondary antibody (1:30,000 dilution; Jackson ImmunoResearch) was added to each well for 30 minutes incubation at room temperature. The plate was washed six times with wash buffer and dried with filter paper. 50 L of TMB peroxidase substrate (SureBlueTM, KPL) was added for 30 minutes reaction and the reaction was stopped by adding 50 L of 2N H.sub.2SO.sub.4. The absorbance at 450 nm was measured by an ELISA reader (Spectra Max M2 model, USA).
1.8 Peptide-ELISA Analysis
[0109] A panel of 165 overlapping synthetic peptides were synthesized using Fmoc chemistry by Kelowna International Scientific Inc. (Taipei Hsien, Taiwan) according to the sequence of VP1 (5715-mer peptides), VP2 (4915-mer peptides), VP3 (4715-mer peptides), and VP4 (1215-mer peptides) capsid proteins of CVA16 [Chong et al, 2012]. Each peptide contained 15 amino acids, 10 residues of which overlapped with the adjacent peptides. The reactivity of the antibody to each synthetic peptide was analyzed by an enzyme-linked immunosorbent assay (ELISA) according to the protocol previously reported by Chong et al. [Chong et al, 2012]. Peptides and viral particles were coated on a 96-well ELISA plate (Corning) at 0.5 g/well in 50 L coating buffer (0.1 M NaHCO.sub.3, pH9.5) [Liu et al., 2016]. After antigen coating, 250 L of 5% skim milk in PBS was added for blocking. Fifty L of primary antibodies were added to testing wells and incubated for 2 hours at room temperature. The wells were washed four times with 250 L wash buffer (PBS+0.05% Tween20), and 100 L of an HRP-conjugated secondary antibody that includes the goat anti-mouse IgG HRP (1:5,000 dilution; GeneTex), the goat anti-rabbit IgG HRP (1:4,000 dilution; Millipore), and anti-chicken IgY HRP (1:20,000 dilution; Sigma Aldrich) were added to each well for 1 hour incubation at room temperature. The plate was washed six times with wash buffer and dried with filter paper. 50 L of TMB peroxidase substrate (eBioscience) was added for 30 minutes reaction and the reaction was stopped by adding 50 L of 2N H.sub.2SO.sub.4. The absorbance at 450 nm was measured by an ELISA reader (Spectra Max M2 model, USA).
1.9 Sequence Alignment and Structure Homology Modeling Prediction
[0110] The genome sequences of all related CVA16 strains were obtained from the NCBI PubMed website (http://www.ncbi.nlm.nih.gov/pubmed/). The genome sequences of the CVA16 P1 gene were aligned using the ClustalW2 program (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The three-dimensional structure of CVA16 (PDB: 6LHA) was used to predict the possible epitope position by homology modeling [He et al., 2020]. The Cn3D v4.3 graphics (http://www.ncbi.nlm.nih.gov) were used to display the position of the identified peptides.
1.10 Ethics Statement
[0111] The animal experiment was conducted in accordance with the guidelines of the Laboratory Animal Center of the National Defense Medical Center (NDMC), Taiwan. The animal use protocols have been reviewed and approved by the NDMC Institutional Animal Care and Use Committee (NDMC-IACUC approval No.: AN-110-16 & AN-111-26).
2. Results
2.1 Preparation of E- and F-CVA16 Viral Particles
[0112] To prepare two types of CVA16 viral particles, the concentrated CVA16 solution were purified using sucrose gradient ultracentrifugation. As shown in
2.2 Production and Characterization of Anti-CVA16 IgYs and Anti-EV-A71 IgY
[0113] To generate anti-CVA16 IgYs and anti-EV-A71 IgY, chickens were immunized with two types of CVA16 viral particles and EV-A71 bulk, respectively. The immunization flow chart was shown in
[0114] To evaluate the antigen recognition of anti-CVA16 IgYs and anti-EV-A71 IgY, three types of antigens (CVA16 E-particle, CVA16 F-particle, and EVA71 bulk) were loaded in SDS-PAGE for western blot assay (
2.4 Reactivity and Specificity of Anti-CVA16 IgYs
[0115] In order to confirm the reactivity and specificity of the anti-CVA16 IgYs generated from CVA16 E-particle and F-particle, three enteroviral particles (CVA16 E-particle, CVA16 F-particle, and EVA71 bulk) were tested in this ELISA analysis. For comparison, the anti-CVA16 IgYs diluted to 110.sup.3 dilutions, which still recognized the EV-A71 particles, is defined as the baseline. The two types of anti-CVA16 IgYs were found to highly recognize CVA16 E-particle and CVA16 F-particle even at 110.sup.5 dilutions. The results show that the anti-CVA16 IgYs have specific reactivity to the CVA16 particles than EV-A71 particles (
2.5 Neutralization of Anti-CVA16 IgYs and Anti-EV-A71 IgY
[0116] Our previous study has shown that the E- and F-particles of CVA16 had profound difference in immunogenicity in mice [Chong et al., 2012]. To investigate whether the E- and F-particles of CVA16 also reveal this variance in chicken model, anti-CVA16-E IgY, anti-CVA16-F IgY and anti-EV-A71 IgY were evaluated by the neutralization assay (Nt titer) against CVA16 and EV-A71 infection, respectively. The results were shown in Table 1. The anti-CVA16-F IgY presented higher neutralizing titers against CVA16 (Nt=5145.31646.5) than the anti-CVA16-E IgY (Nt=354.80). Interesting, the anti-CVA16-E IgY presented cross-neutralizing titers against EV-A71 (Nt=12366.91621.7). However, the anti-CVA16-F IgY failed to neutralize the EV-A71 infection (Nt<16). The anti-EV-A71 IgY samples can neutralize EV-A71 (Nt>524288), but that failed to neutralize CVA16 (Nt<16). Our result indicated that the E- and F-particles of CVA16 should be used as the immunogens for the induction of a good and specific neutralization response against CVA16 in chicken model. The anti-CVA16-E IgY can cross-neutralize the EV-A71. In addition, the anti-EV-A71 IgY cannot cross-neutralize the CVA16.
TABLE-US-00001 TABLE 1 Neutralization titer of anti-CVA16 IgYs and anti-EV-A71 IgY. Sample Virus Anti-CVA16-E Anti-CVA16-F Anti-EV-A71 CVA16 354.8 0 5145.3 1646.5 <16 EV-A71 12366.9 1621.7 <16 >524288 (Nt = Mean SD)
2.6 Liner Epitope Mapping of Anti-CVA16 IgYs and Anti-EV-A71 IgY
[0117] Although current results indicate that both mouse and rabbit antisera have CVA16 virus-specific neutralizing antibodies, it remains worth investigating whether IgY antibodies could cross-react with the reported liner epitopes of CVA16. Our previous study has shown that the antisera of the E- and F-particles of CVA16 induced in mic recognized different liner epitopes [Chong et al., 2012]. As shown in
TABLE-US-00002 TABLE2 Summaryofanti-CVA16IgYsreactedCVA16 syntheticpeptides. Anti- Anti- Aminoacid Peptide CVA16-E CVA16-F SEQIDNO. sequence code IgY IgY SEQIDNO:1 GDPIADMIDQTVNNQ VP1-1 .square-solid. .square-solid. SEQIDNO:2 DMIDQTVNNQVNRSL VP1-2 .square-solid. SEQIDNO:3 TALQVLPTAADTEAS VP1-5 .square-solid. SEQIDNO:4 LPTAADTEASSHRLG VP1-6 .square-solid. SEQIDNO:5 TRCVLNHHSTQETAI VP1-14 .square-solid. SEQIDNO:6 YAQLRRKCELFTYMR VP1-24 .square-solid. SEQIDNO:7 DGYPTFGEHLQANDL VP1-42 .square-solid. .square-solid. SEQIDNO:8 FGEHLQANDLDYGQC VP1-43 .square-solid. SEQIDNO:9 SITLRVYMRIKHVRA VP1-50 .square-solid. SEQIDNO:10 DATAVDKPTRPDVSV VP2-10 .square-solid. SEQIDNO:11 ALLVAVLPEYVLGTI VP2-25 .square-solid. .square-solid. SEQIDNO:12 AGGTGNENSHPPYAT VP2-28 .square-solid. SEQIDNO:13 PYMNTVPFDSALNHC VP2-40 .square-solid. SEQIDNO:14 GIPTELKPGTNQFLT VP3-1 .square-solid. SEQIDNO:15 NQFLTTDDGVSAPIL VP3-3 .square-solid. SEQIDNO:16 TDDGVSAPILPGFHP VP3-4 .square-solid. .square-solid. SEQIDNO:17 SAPILPGFHPTPPIH VP3-5 .square-solid. SEQIDNO:18 PGFHPTPPIHIPGEV VP3-6 .square-solid. SEQIDNO:19 TPPIHIPGEVHNLLE VP3-7 .square-solid. SEQIDNO:20 IPGEVHNLLEICRVE VP3-8 .square-solid. SEQIDNO:21 HNLLEICRVETILEV VP3-9 .square-solid. SEQIDNO:22 MQRLCFPVSVQSKTG VP3-14 .square-solid. SEQIDNO:23 FRADPGRDGPWQSTI VP3-18 .square-solid. SEQIDNO:24 DTEDIEQTANIQ VP3-47 .square-solid. SEQIDNO:25 MSQDPKKFTDPVMDV VP4-10 .square-solid. SEQIDNO:26 KKFTDPVMDVIHEMA VP4-11 .square-solid. SEQIDNO:27 PVMDVIHEMAPPLK VP4-12 .square-solid. .square-solid.: Anti-CVA16 IgY reacted synthetic peptide.
2.7 Potential Location of CVA16 Neutralization Epitopes Estimated by in Silico Simulation
[0118] To define the locations and spatial relationship of these liner epitopes identified in this study. The viral structure model of CVA16 190 strain (PDB: 6LHA) was used as the template for the construction of the molecular structure of CVA16. The structure protein sequences (VP1 to VP4) of the CVA16 5079 strain were aligned using the Clustal W2 program. After labeling these liner epitopes of anti-CVA16 IgYs in structure protein sequences, and the results showed that the VP1-42, VP1-43, VP2-28, and VP3-18 peptides form a region in the canyon at the junction site of VP1, VP2, and VP3 within the capsid (
SEQUENCE INFORMATION
TABLE-US-00003 Strain Sequence Size VP0 CVA16_ MGSQVSTORSGSHENSNSASEGSTINYTTINYYKDAY 323 5079 AASAGRQDMSQDPKKFTDPVMDVIHEMAPPLKSPSAE A.A. ACGYSDRVAQLTIGNSTITTQEAANIVIAYGEWPEYC PDTDATAVDKPTRPDVSVNRFFTLDTKSWAKDSKGWY WKFPDVLTEVGVFGQNAQFHYLYRSGFCVHVQCNASK FHQGALLVAVLPEYVLGTIAGGTGNENSHPPYATTQP GQVGAVLTHPYVLDAGIPLSQLTVCPHQWINLRTNNC ATIIVPYMNTVPFDSALNHCNFGLLVVPVVPLDENAG ATSEIPITVTIAPMCAEFAGLRQAVKQ(SEQID NO:28) VP1 GDPIADMIDQTVNNQVNRSLTALQVLPTAADTEASSH 297 RLGTGVVPALQAAETGASSNASDKNLIETRCVLNHHS A.A. TQETAIGNFFSRAGLVSIITMPTTGTQNTDGYVNWDI DLMGYAQLRRKCELFTYMRFDAEFTFVVAKPNGELVP QLLQYMYVPPGAPKPTSRDSFAWQTATNPSVFVKMTD PPAQVSVPEMSPASAYQWFYDGYPTFGEHLQANDLDY GQCPNNMMGTFSIRTVGTEKSPHSITLRVYMRIKHVR AWIPRPLRNQPYLFKTNPNYKGNDIKCTSTSRDKITT L(SEQIDNO:29) VP2 CVA16_ SPSAEACGYSDRVAQLTIGNSTITTQEAANIVIAYGE 254 5079 WPEYCPDTDATAVDKPTRPDVSVNRFFTLDTKSWAKD A.A. SKGWYWKFPDVLTEVGVFGQNAQFHYLYRSGFCVHVQ CNASKFHQGALLVAVLPEYVLGTIAGGTGNENSHPPY ATTQPGQVGAVLTHPYVLDAGIPLSQLTVCPHQWINL RTNNCATIIVPYMNTVPFDSALNHCNFGLLVVPVVPL DFNAGATSEIPITVTIAPMCAEFAGLRQAVKQ (SEQIDNO:30) VP3 CVA16_ GIPTELKPGTNQFLTTDDGVSAPILPGFHPTPPIHIP 242 5079 GEVHNLLEICRVETILEVNNLKTNETTPMQRLCFPVS A.A. VQSKTGELCAAFRADPGRDGPWQSTILGOLCRYYTQW SGSLEVTFMFAGSFMATGKMLIAYTPPGGNVPADRIT AMLGTHVIWDFGLQSSVTLVVPWISNTHYRAHARAGY FDYYTTGIITIWYQTNYVVPIGAPTTAYIVALAAAQD NFTMKLCKDTEDIEQTANIQ(SEQIDNO:31) VP4 CVA16_ MGSQVSTQRSGSHENSNSASEGSTINYTTINYYKDAY 69 5079 AASAGRQDMSQDPKKFTDPVMDVIHEMAPPLK A.A. (SEQIDNO:32)
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