WO2024229308A1 - THE COMPOSITION OF IMMUNOGLOBULIN IgY IN THE THERAPEUTIC AND PREVENTION OF NOROVIRUS INFECTION - Google Patents

Abstract

The present invention discloses an antibody comprising an immunoglobulin Y (IgY) against the P protein of Human Norovirus (HuNoVs), which can be used for virus detection, treatment, and prevention of infection.

Description

THE COMPOSITION OF IMMUNOGLOBULIN IgY IN THE THERAPEUTIC AND PREVENTION OF NOROVIRUS INFECTION TECHNICAL FIELD OF THE INVENTION ^^{0001^} _{The present invention relates generally to the field of therapeutic and prevention} of norovirus infection, particularly a composition of immunoglobulin Y (IgY) for the therapeutic and prevention of norovirus infection. BACKGROUND OF THE INVENTION ^^{0002^} _{Human noroviruses (HuNoVs) are transmitted through the fecal-oral route,} aerosolized vomitus, contaminated water or food, pollutants, and direct person-to-person contact. They exhibit remarkable persistence in the environment, withstanding freeze-thaw cycles (at least 14 cycles), dry conditions, low pH levels (gastric pH 3-4), and common chemical disinfectants. ^^{0003^} _{Noroviruses (NoVs) belong to the family Caliciviridae, genus Norovirus. NoVs} measure approximately 27-38 nm in size, with a genome length ranging from 7.4 to 7.7 kb. They are non-enveloped viruses, (+)ssRNA viruses. ^^{0004^} _{Among the six genogroups of NoV, Genogroup I and II (GI and GII) are the most} prevalent in human infections. HuNoVs exhibit extensive antigenic and genetic variability. The HuNoVs VP1 capsid folds into two major domains designated S for the shell domain and P for the protruding domain. ^^{0005^} _{The significant antigenic variation between genotypes and genomes of HuNoV} is a major barrier to the development of NoV vaccines. Challenges in vaccine design include complexity, lack of appropriate models, unknown duration of vaccine protection, and difficulty in addressing unknown exposure pathways. In the absence of a vaccine, the only effective methods to mitigate HuNoV outbreaks are prevention, early detection, and control. SUMMARY OF THE INVENTION ^^{0006^} _{Accordingly, one aspect of the invention relates to A antibody comprising an} immunoglobulin (IgY) against the P protein of Human Norovirus (HuNoVs). ^{^0007^} _{In some embodiments, the P protein comprises the amino acid sequence of SEQ} ID NO: 1. ^^{0008^} _{In some embodiments, the Human Norovirus is GI or GII.} ^^{0009^} _{In some embodiments, the antibody is derived from hens.} ^^{0010^} _{Another aspect of the present invention relates to a method to detect the existence} of HuNoVs, comprising contacting the sample with the said IgY antibody. ^^{0011^} _{A further aspect of the present invention relates to a method of treating or} preventing HuNoVs infection in a subject in need thereof, comprising administering to the subject the said IgY antibody. ^^{0012^} _{In some embodiments, the said IgY antibody is deposited in eggs of immunized} hens. ^^{0013^} _{In some preferred embodiments, the said IgY antibody is administered orally.} ^^{0014^} _{In some embodiments, the said IgY antibody binds to human noroviruses,} eliminating them as they pass through the intestinal tract and preventing their replication or transmission. ^^{0015^} _{In some embodiments, the subject is a human.} BREIF DESCRIPTION OF THE DRAWINGS ^^{0016^} _{The detailed description of preferred embodiments of the invention will be more} comprehensible when reviewed alongside the accompanying drawings. The embodiments depicted in the drawings are presently preferred for illustrating the invention. However, it should be noted that the scope of the invention is not confined to the specific arrangements and components illustrated in the embodiments depicted in the drawings. ^^{0017^} Fig. 1 depicts the schematic diagram of production of anti-Norovirus P protein IgY. ^^{0018^} Figs. 2A-2B depict the SDS-PAGE image of anti-Norovirus P protein IgY. ^^{0019^} Fig. 3 depicts the anti Noro-p Protein IgY Ab of yolk. ^{^0020^} _{Fig. 4 depicts the anti Noro-p Protein IgY Ab titer of anti serum and yolk.} ^^{0021^} _{Fig. 5 depicts the schematic diagram of the titration of amount of anti-NoV in}

immunized serum or using PGM assay. ^^{0022^} _{Fig. 6 depicts the results of the titration of amount of anti-NoV in immunized} serum or IgY using PGM assay. DETAILED DESCRIPTION OF THE INVENTION ^^{0023^} _{All technical and scientific terms used herein, unless}

have the same meaning as commonly understood by one of skill in the

in meaning, this specification shall prevail. ^^{0024^} _{The term "antibody," as used herein, refers to an}

_that exhibits specific binding affinity towards an antigen. Antibodies encompass

sourced from natural or recombinant origins, and can also include

from intact immunoglobulins. Antibodies typically exist as tetramers of

to polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)₂

chain antibodies and humanized antibodies. ^^{0025^} _{The term "immunoglobulin," as used herein, refers}

a of as

are linked by a linker, while in dsFvs, the chains have been altered to introduce a disulfide bond for stabilizing their association. The term "immunoglobulin" also encompasses recombinant forms such as chimeric immunoglobulins. ^^{0026^} _{The term "immunoglobulin Y" (IgY), as used herein, refers to a type of} immunoglobulin that is predominantly found in the blood of birds, reptiles, and lungfish. It is also present in significant quantities in chicken egg yolk. Similar to other immunoglobulins, IgY is a class of proteins generated by the immune system in response to specific foreign substances, exhibiting specific recognition of these substances. IgY comprises two light chains and two heavy chains. ^^{0027^} _{The advantages of utilizing IgY antibodies are significant. IgY antibodies can be} stored for extended periods, up to 5-10 years at 4°C, and are suitable for long-term storage at -20°C. They demonstrate remarkable stability across a wide pH range of 4-11. IgY antibodies are accessible in large quantities through non-invasive methods at a low cost, making them practical for various applications. Importantly, IgY does not trigger the human complement system or interact with Fc receptors, thereby minimizing the risk of inflammatory responses. Additionally, IgY exhibits lower cross-reactivity with mammalian IgG compared to traditional antibodies, reducing the likelihood of false positive test results. Due to the strong immunogenicity of mammalian proteins in birds, IgY is particularly effective for immune responses against avian antigens. These advantages collectively position IgY as a valuable tool in biomedical research, diagnostics, and therapeutic applications. Example 1 ^^{0028^} _{The experimental protocol involves administering D(0) intramuscular (IM)} injections of P protein (P1+P2) at a dosage of 80 μg per dose. Subsequently, after 1 month post- immunization (D(1M)), another intramuscular injections of P protein are administered. Following this, starting from day 2 months post-immunization (D(2M)) and continuing for 52 weeks, the hens are expected to lay approximately 5 eggs per week, resulting in an estimated yield of around 250 eggs per year (see Fig. 1). ^^{0029^} _{The SDS-PAGE image of anti-Norovirus P protein IgY is shown in Figs.2A (No.} 51) and 2B (No.52), and the concentration of the anti-Norovirus P protein IgY is detailed in Table 1. Table 1 ch C 50 To

^^{0030^} _{The anti Noro-P protein IgY Ab of yolk is shown in Fig. 3 and Table 2. The titer} of anti Norovirus P protein IgY Ab of chicken anti serum and yolk are shown in Fig. 4, Table 3 and Table 4. Table 2 ^{co co co co}

Table 3 N an yo

g . . . . No

rovirus P No.52 D0 D15 D30 D45 D60 anti serum I Y <100 1E+04 1 E+05 1 E+06 1E+06 yo

Example 3 ^^{0031^} _{The schematic diagram of the titration of amount of anti-NoV in immunized} serum or IgY using PGM assay is shown in Fig. 5. The results are shown in Fig. 6 and Table 5. The OD450 of PBS control (no antibody) for EL51 and EL52 are 0.606 and 0.593 respectively. The dilution set of the sample eliciting >50% inhibition of OD450 nm obtained from PBS control. EL51 IgY IC50: dilution at 320 fold, 17.9/320 = 56 ng/μL. EL52 IgY IC50: dilution at 320 fold 19.6/320 = Table 5 d E E D E E

disclosure and is not intended to limit the scope of the present invention. Any changes and modifications in the concentration, temperature, pH, reaction time and spirits mentioned in the scope of the patent application shall be included in the scope of the patent application for this work.

Claims

C L A I M S 1. A antibody comprising an immunoglobulin (IgY) against the P protein of Human Norovirus (HuNoVs). 2. The antibody of claim 1, wherein the P protein comprises the amino acid sequence of SEQ ID NO: 1. 3. The antibody of claim 1, wherein the Human Norovirus is GI or GII. 4. The antibody of claim 1, wherein the antibody is derived from hens. 5. A method to detect the existence of HuNoVs, comprising contacting the sample with the antibody of claim 1. 6. A method of treating or preventing HuNoVs infection in a subject in need thereof, comprising administering to the subject the antibody of claim 1. 7. The method of claim 6, wherein the antibody is deposited in eggs of immunized hens. 8. The method of claim 7, wherein the antibody is administered orally. 9. The method of claim 8, wherein the antibody binds to human noroviruses, eliminating them as they pass through the intestinal tract and preventing their replication or transmission. 10. The method of claim 6, wherein the subject is a human.