Epitope tuning controls antibody specificity in polyclonal humoral responses

Abstract

In alternative embodiments, chimeric immunogens and methods for their preparation and use are provided, including methods for preparing and obtaining polyclonal antibodies specific to selected epitopes. In alternative embodiments, methods are provided for generating a balanced immune response against multiple epitopes present in an antigen, the methods comprising immunizing a host with multiple polypeptides, wherein each polypeptide contains one or a subset of the multiple epitopes. In alternative embodiments, methods are provided for generating a balanced epitope-specific antibody response in a non-human mammalian host, wherein the immune response includes generating a host antibody specifically targeting (or specifically binding to) at least one human epitope, and the methods include administering a sufficient amount of the chimeric or recombinant polypeptide to the host to generate the epitope-specific antibody response.

Description

[0001] Related applications This Patent Cooperation Treaty (PCT) international application claims priority to U.S. Provisional Patent Application Serial No. 63 / 617,927, filed January 5, 2024, pursuant to 35 USC §119(e). The entire contents of the aforementioned application are expressly incorporated herein by reference for all purposes. All publications, patents, and patent applications cited herein are expressly incorporated herein by reference for all purposes.

[0002] Reference to the electronic sequence list This application contains a sequence list, which has been electronically submitted in XML format, the entire contents of which are incorporated herein by reference. The XML copy was created on December 21, 2024, and is named “6363.149133PCT.xml” with a size of 16,128 bytes. The sequence list contained in this XML file is part of this specification, and the entire contents are incorporated herein by reference. Technical Field

[0003] This invention generally relates to immunology and immunoassay. In alternative embodiments, chimeric immunogens and methods for their preparation and use are provided, including methods for preparing and obtaining polyclonal antibodies specific to selected epitopes. In alternative embodiments, methods are provided for generating a balanced immune response against multiple epitopes present in an antigen, the method comprising immunizing a host with multiple polypeptides, wherein each polypeptide contains one or a subset of the multiple epitopes. In alternative embodiments, methods are provided for generating a balanced epitope-specific antibody response in a non-human mammalian host, wherein the immune response comprises generating a host antibody specifically targeting (or specifically binding to) at least one human epitope, and the method comprises administering a sufficient amount of the chimeric or recombinant polypeptide to the host to generate the epitope-specific antibody response. Background Technology

[0004] Polyclonal antibodies exhibit diverse reactivity to multiple epitopes, ensuring robust responses even in the face of target diversity or environmental changes. To obtain polyclonal antibodies, animals are first immunized with proteins, protein fragments, or mixtures thereof. The humoral immune system then selects antibody-producing B cell clones for expansion and maturation. In later stages, these B cells further diversify through mutagenesis and selection of high-affinity immunoglobulin genes. While the immune system possesses the fundamental ability to produce antibodies against virtually any exogenous protein, certain epitopes are known to be dominant, and B cell clones that produce antibodies recognizing these “dominant” epitopes will take over the immune response. This means that standard polyclonal antibodies are biased towards some epitopes and may lack responsiveness to others.

[0005] In principle, immunization can be performed using a single (e.g., linear) epitope, a single peptide, or a mixture of peptides. However, peptides may not have the same three-dimensional (3D) structure as the proteins from which they are derived, resulting in the generation of antibodies with low or no affinity for said proteins. Peptides (especially those that are non-dominant epitopes) are often too small to spontaneously trigger an immune response and require either being constructed into larger structures or relying on co-stimulation with more immunogenic components to stimulate the immunized animal to generate antibodies against epitopes other than those favored by humoral responses.

[0006] Various techniques, such as neonatal drug-induced masking subtractive immunization or high-band tolerance methods (see, for example, U.S. Patent 7,598,030; U.S. Patent 8,133,744), can be used to induce tolerance to unselected epitopes. However, tolerance can be a flawed process, where antibody clones targeting unselected epitopes persist at a certain level; and combinations have been suggested for greater efficiency. In all cases, tolerance implies the need for additional procedures as part of the process in addition to standard immunization, thus increasing complexity and cost.

[0007] Antibodies used for commercial purposes are purified from the serum of immunized animals. Even if total immunoglobulins can be extracted, antibodies often need further purification by removing unwanted reactivity (adsorption purification) or by specifically selecting the desired reactivity (affinity purification).

[0008] It will be advantageous to be able to specify which particular epitopes the polyclonal antibody will recognize without adding additional steps to the immunization and / or purification process. For example, it will be advantageous to eliminate the need for expensive and time-consuming adsorption and / or affinity purification steps. Summary of the Invention

[0009] In alternative embodiments, methods and compositions are provided for generating a balanced immune response against multiple epitopes present in an antigen, the methods comprising immunizing a host with multiple polypeptides, wherein each polypeptide comprises one of the multiple epitopes or a subset of the multiple epitopes.

[0010] In alternative implementations of the methods provided herein: - A balanced polyclonal antibody reaction is a polyclonal antibody reaction that includes (or results in) multiple polyclonal antibody groups, each of which can specifically bind to a different epitope on an antigen, and none of the epitopes is significantly dominant over the others, thereby producing a balanced polyclonal antibody reaction or a balanced polyclonal serum. - A balanced polyclonal antibody reaction or balanced polyclonal serum contains multiple antibody groups with roughly similar titers; - In the polyclonal reaction, none of the polyclonal antibody groups has about 5%, 10%, 20%, or 30% more antibodies than any other polyclonal antibody group in the plurality of polyclonal antibody groups; - In the polyclonal reaction, none of the plurality of polyclonal antibody groups has a signal strength, signal-to-noise ratio, or signal rate that is about 5%, 10%, 20%, or 30% or more greater than any other polyclonal antibody group in the plurality of polyclonal antibody groups; - The fact that no single epitope is significantly superior to the others implies that each antibody swarm has a higher binding affinity, or affinity constant K, to its corresponding antigen. a and / or dissociation constant K d Compared to all other antibody groups, the difference is no greater than approximately 5%, 10%, 20%, or 30% higher or lower; The method further includes: identifying whether one or more dominant epitopes exist among the plurality of epitopes; and optionally, the one or more dominant epitopes are identified by: immunizing a first species with the antigen and identifying a polyclonal antibody group generated in the first species and an epitope binding to the polyclonal antibody group, and determining whether one or more epitopes or polyclonal antibody groups are dominant compared to other epitopes or polyclonal antibody groups; and optionally, if the antibody group binding to the first epitope has a titer for the first epitope that is at least about 5%, 10%, 20%, or 30% higher binding affinity to the corresponding epitope than other antibody groups, then the first epitope is considered dominant compared to the other epitopes; - The method further includes engineering the modified antigen by: removing one, several, or all of the identified one or more dominant epitopes from the antigen, or modifying the structure of the antigen responsible for creating the one or more dominant epitopes such that the one or more dominant epitopes are no longer immunogenic in the first species, or have significantly lower immunogenicity in the first species, and optionally, significantly lower immunogenicity means immunogenicity that is at least about 85%, 90%, or 95% lower; -The method further includes isolating or substantially purifying polyclonal antibodies against an immune host; - Antigens containing multiple epitopes include proteins, optionally recombinant chimeric proteins; -The recombinant chimeric antigen polypeptide containing multiple epitopes comprises: (a) Polypeptides derived from the first species, and (b) at least one epitope comprising a heterologous amino acid sequence or amino acid residue derived from at least a second species. At least one heterologous amino acid sequence or amino acid residue derived from the second species is inserted into, linked to, created in, or replaces or substitutes the amino acid sequence of the polypeptide derived from the first species; Furthermore, the amino acid sequence of the recombinant chimeric antigen polypeptide is essentially composed of amino acid sequences derived from the first species. Furthermore, when the amino acid sequence from the second species is inserted into, linked to, created in, or replaces or substitutes for a portion of the amino acid sequence of the polypeptide from the first species, at least one new epitope is generated, formed, or created on the polypeptide from the first species. When the recombinant chimeric antigen polypeptide is applied to the first species, the new epitope enables the first species to generate a humoral antibody response specific to the at least one new epitope. When the recombinant chimeric antigen polypeptide is used to generate a humoral immune response from an animal of the first species, the polyclonal antibody generated in the first species binds substantially only specifically to the at least one novel epitope and substantially nonspecifically to a polypeptide derived from the first species that lacks the at least one novel epitope, or an epitope created, formed, or generated by the at least one heterologous amino acid sequence or amino acid residue derived from the second or additional species, wherein the at least one heterologous amino acid sequence or amino acid residue is inserted into, linked to, created in, or replaces or substitutes the portion of the polypeptide derived from the first species.

[0011] - The recombinant chimeric antigen polypeptide containing multiple epitopes derived from the second species is a homolog of the polypeptide derived from the first species; - An amino acid sequence from at least one second species is homologous to the first species and is inserted into a portion of the amino acid sequence of the polypeptide derived from the first species, linked to the portion, created in the portion, or replaces or substitutes the portion of the at least one homologous second species sequence, replacing all or almost all structurally homologous segments or portions of the amino acid sequence of the polypeptide derived from the first species. - An amino acid sequence from at least one second species is homologous to the first species and is inserted into, linked to, created in, or replaces the portion of the amino acid sequence of the polypeptide derived from the first species. The at least one homologous second species sequence is structurally homologous to the amino acid sequence of the polypeptide derived from the first species. - The homologs of the first species have at least about 25% to 99% sequence identity with their homologs in the second species; -The homologs of the first species have substantially the same secondary and / or tertiary structures as their homologs in the second species; - The homolog of the first species has at least about 25% to 99% sequence identity with its homolog in the second species, and has substantially the same secondary and / or tertiary structure as its homolog in the second species; - The homolog of the first species has at least about 50% sequence identity with its homolog in the second species, or the homolog of the first species has at least about 70% sequence identity with its homolog in the second species, or the homolog of the first species has at least about 80% sequence identity with its homolog in the second species, or the homolog of the first species has at least about 90% sequence identity with its homolog in the second species. - The amino acid sequences from the first species and the amino acid sequences from the second species have Z scores of approximately 2 to approximately 8 when aligned using distance matrix alignment; - The amino acid sequences from the first species and the amino acid sequences from the second species have a Z score of at least 8 when aligned using distance matrix alignment; -Antibodies are polypeptides derived from the first species and their homologous polypeptides derived from the second species; - The polypeptide derived from the first species and at least one heterologous amino acid sequence derived from the second species are both derived from the antibody heavy chain or antibody light chain. - The antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain, or the light chain is a κ or λ light chain; - The first species is a mammal species; the second animal is a mammal species; or, the first species is a Galliformes (Gallusia). Galliformes ) or Pheasant family ( Phasianidae The first species is an animal, and the second species is a mammal species; or the first species is a rabbit, a rodent species, a sheep, a goat, a pig, a cow, a horse, or a chicken; and the second species is a human, or the rodent species is a rat or a mouse. - At least about 80% to about 99% of the amino acid sequence of the recombinant chimeric antigen polypeptide is derived from the amino acid sequence of the first species, and / or about 1% to about 20% of the amino acid sequence of the recombinant chimeric antigen polypeptide is derived from the amino acid sequence of the at least one second species. - One, two, three, four, five, six, seven, eight or more dominant epitopes are removed or deleted, or modified so that they are no longer immunogenic in the first species, or their immunogenicity in the first species is significantly reduced; - The recombinant chimeric antigen polypeptide is prepared by a method further comprising: removing the one or more new epitopes from at least one heterologous amino acid sequence or amino acid residues derived from the second species or an additional species after inserting one or more new epitopes into a portion of the amino acid sequence of the polypeptide derived from the first species, linking to the portion, creating in the portion, or replacing or substituting the portion. - Inserting at least two or more different heterologous amino acid sequences or amino acid residues into a portion of the amino acid sequence of the polypeptide derived from the first species, linking to the portion, creating in the portion, or replacing or substituting the portion; - The at least two or more different heterologous amino acid sequences or amino acid residues are from different animal species, or at least one of the at least two or more different heterologous amino acid sequences or amino acid residues is from humans, and at least one of the at least two or more different heterologous amino acid sequences or amino acid residues is from non-human or animal species. - At least one of the heterologous amino acid sequences or amino acid residues contains an artificial epitope that is not derived from the at least one second species; - At least one of the heterologous amino acid sequences or amino acid residues contains an epitope that is originally derived from the at least one second species and is immune-silenced in the first species (cannot generate an antibody response in the first species), but is modified to become an immune-active epitope that can generate an antibody response against it by the first species. - At least one novel epitope in the heterologous amino acid sequence or amino acid residues is modified such that the antibody generated by the first species against the modified novel epitope binds with lower strength or slower compared to a comparable unmodified novel epitope; - The recombinant chimeric antigen polypeptide further comprises at least one novel epitope derived from at least a second species, which is not derived from the first species, and the at least one novel epitope enables the generation of antibodies against it in the first species; A portion of the multiple polypeptides comprises an amino acid sequence from the same species as the host, and the multiple epitopes originate from a different species than the host, and optionally, the host produces antibodies against each of the multiple epitopes in substantially similar titers; and / or - The portions of the multiple polypeptides are derived from rabbits, and the multiple epitopes are derived from species other than rabbits, and optionally, the portions of the multiple polypeptides are derived from rabbit CDv6 polypeptide.

[0012] In an alternative embodiment, a composition comprising a plurality of polypeptides is provided, wherein each polypeptide comprises one or a subset of the plurality of epitopes present in the antigen.

[0013] In alternative embodiments of the compositions provided herein: - At least one of the multiple polypeptides is engineered to remove one, several or all of the dominant epitopes from the antigen, or has been modified such that one or more dominant epitopes are no longer immunogenic in the first species, or are significantly reduced in immunogenicity in the first species. - A significant reduction in immunogenicity means a reduction in immunogenicity of at least approximately 85%, 90%, or 95%; - Antigens containing multiple epitopes include proteins, optionally recombinant chimeric proteins; -The recombinant chimeric antigen polypeptide containing multiple epitopes comprises: (a) Polypeptides derived from the first species, and (b) at least one epitope comprising a heterologous amino acid sequence or amino acid residue derived from at least a second species. At least one heterologous amino acid sequence or amino acid residue derived from the second species is inserted into, linked to, created in, or replaces or substitutes the amino acid sequence of the polypeptide derived from the first species; Furthermore, the amino acid sequence of the recombinant chimeric antigen polypeptide is essentially composed of amino acid sequences derived from the first species. Furthermore, when the amino acid sequence from the second species is inserted into, linked to, created in, or replaces or substitutes for a portion of the amino acid sequence of the polypeptide from the first species, at least one new epitope is generated, formed, or created on the polypeptide from the first species. When the recombinant chimeric antigen polypeptide is applied to the first species, the new epitope enables the first species to generate a humoral antibody response specific to the at least one new epitope. When the recombinant chimeric antigen polypeptide is used to generate a humoral immune response from an animal of the first species, the polyclonal antibody generated in the first species binds substantially only specifically to the at least one novel epitope and substantially nonspecifically to a polypeptide derived from the first species that lacks the at least one novel epitope, or an epitope created, formed, or generated from the at least one heterologous amino acid sequence or amino acid residue derived from the second or additional species, wherein the at least one heterologous amino acid sequence or amino acid residue is inserted into, linked to, created in, or replaces or substitutes the portion of the polypeptide derived from the first species; - The recombinant chimeric antigen polypeptide containing multiple epitopes derived from the second species is a homolog of the polypeptide derived from the first species; - An amino acid sequence from at least one second species is homologous to the first species and is inserted into a portion of the amino acid sequence of the polypeptide derived from the first species, linked to the portion, created in the portion, or replaces or substitutes the portion of the at least one homologous second species sequence, replacing all or almost all structurally homologous segments or portions of the amino acid sequence of the polypeptide derived from the first species. - An amino acid sequence from at least one second species is homologous to the first species and is inserted into, linked to, created in, or replaces the portion of the amino acid sequence of the polypeptide derived from the first species. The at least one homologous second species sequence is structurally homologous to the amino acid sequence of the polypeptide derived from the first species. - The homologs of the first species have at least about 25% to 99% sequence identity with their homologs in the second species; -The homologs of the first species have substantially the same secondary and / or tertiary structures as their homologs in the second species; - The homolog of the first species has at least about 25% to 99% sequence identity with its homolog in the second species, and has substantially the same secondary and / or tertiary structure as its homolog in the second species; - The homolog of the first species has at least about 50% sequence identity with its homolog in the second species; or the homolog of the first species has at least about 70% sequence identity with its homolog in the second species; or the homolog of the first species has at least about 80% sequence identity with its homolog in the second species; or the homolog of the first species has at least about 90% sequence identity with its homolog in the second species. - When the comparison is performed using distance matrix alignment, the Z score of the amino acid sequence from the first species and the amino acid sequence from the second species is about 2 to about 8, or when the comparison is performed using distance matrix alignment, the Z score of the amino acid sequence from the first species and the amino acid sequence from the second species is at least 8; -Antibodies are polypeptides derived from the first species and their homologous polypeptides derived from the second species; - The polypeptide derived from the first species and at least one heterologous amino acid sequence derived from the second species are both derived from the antibody heavy chain or antibody light chain. - The antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain, or the light chain is a κ or λ light chain; - The first species is a mammal species; the second animal is a mammal species; or, the first species is a Galliformes (Gallusia). Galliformes ) or Pheasant family ( Phasianidae The first species is an animal, and the second species is a mammal species, or the first species is a rabbit, a rodent species, a sheep, a goat, a pig, a cow, a horse, or a chicken; and the second species is a human, or the rodent species is a rat or a mouse.

[0014] - At least about 80% to about 99% of the amino acid sequence of the recombinant chimeric antigen polypeptide is derived from the amino acid sequence of the first species, and / or about 1% to about 20% of the amino acid sequence of the recombinant chimeric antigen polypeptide is derived from the amino acid sequence of the at least one second species. - One, two, three, four, five, six, seven, eight or more dominant epitopes are removed or deleted, or modified so that they are no longer immunogenic in the first species, or their immunogenicity in the first species is significantly reduced; - After inserting one or more new epitopes into a portion of the amino acid sequence of the polypeptide derived from the first species, linking to the portion, creating in the portion, or replacing or substituting the portion, the one or more new epitopes have been removed from at least one heterologous amino acid sequence or amino acid residue derived from the second species or an additional species; - At least two or more different heterologous amino acid sequences or amino acid residues have been inserted into, linked to, created in, replaced or superseded a portion of the amino acid sequence of the polypeptide derived from the first species; - The at least two or more different heterologous amino acid sequences or amino acid residues are from different animal species; - At least one of the at least two or more different heterologous amino acid sequences or amino acid residues is derived from humans, and at least one of the at least two or more different heterologous amino acid sequences or amino acid residues is derived from non-human or animal species. - At least one of the heterologous amino acid sequences or amino acid residues contains an artificial epitope that is not derived from the at least one second species; - At least one of the heterologous amino acid sequences or amino acid residues contains an epitope that is originally derived from the at least one second species and is immune-silenced in the first species (cannot generate an antibody response in the first species), but is modified to become an immune-active epitope that can generate an antibody response against it by the first species. - At least one novel epitope in the heterologous amino acid sequence or amino acid residues is modified such that the antibody generated by the first species against the modified novel epitope binds with lower strength or slower compared to a comparable unmodified novel epitope; - The recombinant chimeric antigen polypeptide further comprises at least one novel epitope derived from at least a second species, which is not derived from the first species, and the at least one novel epitope enables the generation of antibodies against it in the first species; A portion of the multiple polypeptides comprises an amino acid sequence from the same species as the host, and the multiple epitopes originate from a different species than the host, and optionally, the host produces antibodies against each of the multiple epitopes in substantially similar titers; and / or - The portions of the multiple polypeptides are derived from rabbits, and the multiple epitopes are derived from species other than rabbits, or the portions of the multiple polypeptides are derived from the rabbit CDv6 polypeptide, or the portions of the multiple polypeptides are derived from the rabbit myoglobin polypeptide.

[0015] Details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will become apparent from the specification, drawings, and claims.

[0016] All publications, patents, and patent applications cited in this article are expressly incorporated herein by reference in their entirety for all purposes. Attached Figure Description

[0017] The patent or application documents contain at least one color drawing. A copy of the color drawing disclosed in this patent or patent application will be provided by the patent office upon request and payment of the necessary fees.

[0018] The accompanying drawings are illustrations of exemplary embodiments provided herein and are not intended to limit the scope of the invention as covered by the claims.

[0019] Figure 1 An exemplary immunization method or protocol using an antigen having a number of human epitopes is schematically illustrated, as detailed in Example 1 below.

[0020] Figure 2 The results of immunization using the exemplary immunization method are illustrated graphically, as detailed in Example 1 below.

[0021] Figures 3A to 3G The data were graphically presented to demonstrate the superiority of using a single epitope construct for immunization: the responsiveness of four different immunizations to five putative epitopes was compared. Among them: Figure 3A Regarding Ep1, Figure 3B Regarding Ep2, Figure 3C Regarding Ep3, Figure 3C For Ep4, and Figure 3E Regarding Ep5, Figure 3FThe diagram schematically illustrates all epitopes identified by ELISA, as well as Cdv6, indicating that the rabbit λ light chain constant structural domain carries all five human epitopes; and Figure 3G The chimeric Cdv6 structural domain is schematically illustrated, with five positions labeled 1 to 5 and represented by different colors, and highlighted by circles; and the structure is rotated 180 degrees to illustrate position 5. As detailed in Example 1 below.

[0022] Figure 4 The sequence and structure of myoglobin (excluding heme group) are illustrated: six chimeric constructs are shown, each carrying a human epitope in a rabbit background (rabbit sequence is (SEQ ID NO:10)); numbers indicate predicted human epitopes 1 to 6 (human sequence is (SEQ ID NO:11)); as detailed in Example 2 below.

[0023] Figure 5 An exemplary immunization method or protocol is schematically illustrated in which rabbit groups (5 rabbits per group) are immunized with an equal amount of protein (100 µg / injection) of natural human myoglobin immunogen (Myo) containing all epitopes, or a mixed pool of chimeric immunogens (20 µg each of Ep1, Ep2, Ep3, Ep4 and Ep5), wherein a single epitope is grafted into the rabbit myoglobin cassette, as detailed in Example 3 below.

[0024] Figures 6A to 6C Images illustrating SDS-PAGE purity and Western blot analysis are shown below: Figure 6A Images of SDS and protein blots are shown, where the purified protein sample appears as a weak band in lane 1 of the SDS gel (Ep1, first purification; not observed in lane 2, Ep1, second purification), and no additional bands are observed in any of the other lanes (lanes 3, 4, 5, 6, and 7 correspond to Ep2, Ep3, Ep4, Ep5, and human myoglobin, respectively); and Figure 6B Images of protein blots were plotted, showing anti-E. coli ( E. coli The experiment investigated whether impurities related to the expression system still existed: only lane 1 (Ep1 first purification and lane 6 human myoglobin) contained weak bands indicating some impurities; Figure 6C Images of a Western blot experiment using anti-human myoglobin are shown: only lane 1 has a different size than myoglobin, and the second purification of Ep1 (lane 7, ... Figure 6B Excess bands were removed while retaining Ep1 myoglobin (lane 7, Figure 6C(This is described in detail in Example 3 below.) As detailed in Example 3 below.

[0025] Figure 7 The UV-Vis spectra, measured to verify the expression and purification of recombinant holomyoglobin, are plotted graphically: the Solvay band near 409 nm indicates that all recombinant myoglobin variants are loaded with heme groups, and the data indicate that all recombinant myoglobins are in the heme-carrying state. 3+ Oxidized methemoglobin; and the Solvay band of natural human myoglobin is located around 418-422 nm, indicating that it is in the state of oxymyoglobin or carboxymyoglobin, and Table 1 (see below, Example 3) shows the measurement results of five limiting values, as detailed in Example 3 below.

[0026] Figure 8 The results of immunization using the exemplary immunization method are illustrated graphically, as detailed in Example 3 below.

[0027] Figure 9 The structure of rabbit myoglobin is illustrated graphically, in which five human epitopes replace the corresponding rabbit amino acids, as detailed in Example 3 below.

[0028] Figure 10 The reactivity of Group 1 and Group 2 to various coatings was graphically illustrated: the immune responses of two different immunizations using either an Epx box grafted with five different epitopes or natural human myoglobin (hMyo) were assessed using different coatings, and EC50 values ​​were estimated by curve fitting. ELISA data (labels) and curve fittings (lines) are detailed in Example 3 below.

[0029] Figure 11 Table 2 illustrates the EC50 values ​​calculated via curve fitting. Two pools of antiserum from two groups of four rabbits immunized with either an epitope construct mixture (Group 1) or natural human myoglobin (Group 2) were used as the primary antiserum in ELISA experiments to evaluate the immunization strategy. EC50 values ​​were estimated via curve fitting and used to calculate the Group 1 / Group 2 ratio to understand the order of improvement, as detailed in Example 3 below.

[0030] Figures 12A to 12F The development of the immune response over time is illustrated: antiserum blood samples (sample 1 - line and sample 2 black solid line) from four individual rabbits (1, 2, 3, and 4) from two groups (1 and 2, Epx and hMyo, respectively) were analyzed by ELISA to track the immune response against the following substances: Figure 12A Episode 1 (Ep1), Group 1 Figure 12B Episode 1 (Ep1) Group 2 Figure 12C Epitope 3 (Ep3), Group 1 Figure 12D Epitope 3 (Ep1), Group 2 Figure 12E Natural human myoglobin (hMyo), carrying all epitopes, Group 1. Figure 12F Natural human myoglobin (hMyo), carrying all epitopes, Group 2. Furthermore, all data were fitted to find EC50 values ​​(Table 3). Figure 13 The EC50 value was used for a paired t-test to evaluate the two immunization strategies.

[0031] Figure 13 Table 3 shows the EC50 values ​​calculated by curve fitting of the data, as shown in Figure 12.

[0032] Figures 14A to 14F The development of the immune response over time is illustrated; antiserum blood samples (sampling 1 - line and sampling 2 black solid line) from four individual rabbits (1, 2, 3, and 4) from two groups (1 and 2, Epx and hMyo, respectively) were analyzed by ELISA to track the immune response against the following substances: Figure 14A Epitope 2 (Ep2), Group 1, Figure 14B Epitope 2 (Ep2), Group 2 Figure 14C Epitope 4 (Ep4), Group 1 Figure 14D Epitope 4 (Ep4), Group 2 Figure 14E Epitope 5 (Ep5), Group 1 Figure 14F Epitope 5 (Ep5), Group 2 All data were fitted to find EC50 values ​​(Table 4). Figure 15 The EC50 value was used for a paired t-test to compare the two immunization strategies.

[0033] Figure 15Table 4 illustrates that there was no significant increase in antibody titers against Ep2, Ep4, or Ep5 between the first and second blood samples; paired t-tests were used to analyze both the Epx and hMyo groups; in all cases, the EC50 values ​​were not statistically different from the null hypothesis (the difference between the means of the first and second blood samples was zero); therefore, this test could not confirm that additional immunization led to an increase in titers against epitopes 2, 4, or 5 in either group.

[0034] Figure 16 Table 5 illustrates that immunization with the epitope mixture (Epx) resulted in a significant increase in antibody titers against both Ep1 and Ep3; the EC50 values ​​used for statistical analysis are from Table 3 ( Figure 13 ) and Table 4 ( Figure 15 All four animals immunized with Epx responded, and paired t-tests showed a statistically significant difference between using Epx and human myoglobin as coatings in the ELISA test. *Three of the four rabbits showed an increase in titer from the first to the second blood sample,** (n=3; non-responders were excluded from the analysis),***only one of the four rabbits showed an increase in titer from the first to the second blood sample.

[0035] Figure 17 The progression was graphically illustrated as the current titer increases and the progress slows down: further analysis was performed on rabbits immunized with Epx to understand the trend of titer development; the ratio of the EC50 value of the first blood sample to the EC50 value of the second blood sample to the EC50 value of the first blood sample was plotted; the trend appears to be that the higher the current titer, the less the titer increases, or even the lower it may be; this trend suggests that the difference in antibody titers against epitopes decreases over time; Ep1: triangle; Ep2: square; Ep3: circle; Ep4: rhombus; Ep5: line.

[0036] Figures 18A to 18C The illustration schematically depicts how antibodies cover more than one epitope and thus create spatial constraints during epitope binding: Figure 18A Comparison of the size of human myoglobin (surface shown in gray, epitopes shown in color, PDB entry: 3rgk) and human IgG (heavy chain gray and light chain pale yellow, PDB entry: 1hzh); Figure 18B The surface representation of human myoglobin shows the locations of epitopes 2 (green), 3 (sky blue), and 5 (dark red). The spatial arrangement of these three epitopes could potentially weaken the immune response due to structural obstruction, which could be addressed by immunizing with cassettes carrying individual epitopes; and Figure 18CThis diagram illustrates an example of structural barriers, where complementary sites potentially create structural barriers against epitopes 2 and 5 during binding with epitope 3. Atoms in the epitopes are colored according to element type: oxygen (red), nitrogen (blue), and sulfur (yellow).

[0037] The same reference symbols in each figure represent the same element. Detailed Implementation

[0038] In alternative embodiments, chimeric immunogens and methods for their preparation and use are provided, including methods for preparing and obtaining polyclonal antibodies specific to selected epitopes. In alternative embodiments, methods are provided for generating a balanced immune response against multiple epitopes present in an antigen, the methods comprising immunizing a host with multiple polypeptides, wherein each polypeptide contains one epitope or a subset of the multiple epitopes.

[0039] In alternative embodiments, the methods for generating balanced immune responses, as provided herein, address the problem that bias in antibody responses to antigenic epitopes reduces the effectiveness of polyclonal antibody responses for assay development and use. When using polyclonal antibodies for agglutination reactions, the repertoire should be diverse to form large and complex structures. If the polyclonal antibody reactivity is biased towards one or a few epitopes, agglutination may be less pronounced, thus reducing the sensitivity of assays based on such agglutination. Balanced immune responses, as provided herein, address these issues.

[0040] The balanced immune response presented in this article also addresses the problem of selecting immunized animals with the desired response, but then discarding these animals later in the immunization program, resulting in significant costs.

[0041] Furthermore, by using a balanced immune response as described herein, polyclonal antibodies can be manipulated without selectively removing or adding antibodies targeting certain epitopes, thus saving processing time and costs.

[0042] The problems addressed by using the balanced immune response approach presented in this article include: Immunization with natural antigens can lead to antibody library bias. This inferior antibody composition results in a loss of signal intensity (reduced absorbance or light scattering) in agglutination assays. Using a balanced reaction method as presented herein ensures a more uniform distribution of the antibody population.

[0043] Standardization of polyclonal antibodies may require selecting antibody-producing animals, leading to increased costs. Using a balanced reaction method as presented in this paper ensures more uniform titers within the animal, enabling the utilization of most of the antibody and avoiding loss.

[0044] Standardization of polyclonal antibodies may require altering antibody composition by reducing or increasing specific reactivity, which can lead to increased manufacturing time and costs. Using a balanced immune response approach, as presented herein, ensures a more consistent composition of polyclonal antibodies in most animals, thereby eliminating or significantly reducing the need for reprocessing polyclonal antibodies.

[0045] The advantages of using the balanced immunization approach as described herein include, for example: ensuring that all epitopes are available without interference; ensuring that all epitopes generate useful levels of antibodies; and ensuring that most immunized animals are available for antibody production.

[0046] Chimeric or recombinant peptides and nucleic acids In alternative embodiments, chimeric or recombinant polypeptides and methods for their preparation and use are provided. In alternative embodiments, chimeric or recombinant nucleic acids encoding and expressing polypeptides as provided herein are provided, including expression vectors containing and expressing these nucleic acids, cells containing and expressing these nucleic acids, and whole-organism expression systems.

[0047] In alternative embodiments, the recombinant peptides provided herein can be prepared and expressed using any method known in the art, including, for example, using whole organisms such as fungi, plants, or animals (such as mice), and cell cultures derived from whole organisms (such as cultured mammalian cells), or using single-celled organisms such as algae, fungi, yeast, insects (e.g., baculoviruses), or bacterial cells.

[0048] The choice of organisms used to produce (e.g., recombinantly generate) chimeric or recombinant peptides and / or nucleic acids as provided herein can depend on several factors, including whether secondary modifications such as glycosylation are desired or required, or whether protein association with or insertion into a membrane system (e.g., in situ) is desired or required, or whether a specific protein folding pattern is desired or required, and / or whether disulfide bridge formation is desired or required.

[0049] In alternative embodiments, the nucleic acid for expressing the chimeric or recombinant polypeptide as provided herein, such as for in vitro or in vivo expression, is contained in an expression vector, such as in an expression cassette, vector, recombinant virus, artificial chromosome, viscera, or plasmid. In alternative embodiments, the nucleic acid or expression vector expressing the chimeric or recombinant polypeptide as provided herein is administered to an animal (e.g., as naked DNA, which may be suitably formulated) with the aim of inducing a humoral immune response in the animal against the epitopes in the recombinant polypeptide as provided herein.

[0050] In alternative embodiments, a protein-coding DNA sequence (which may be present in an expression vector) is transferred into an organism or cell and placed under the control of relevant expression elements, such as transcription promoters, enhancers, and / or polyadenylation signaling sequences. In alternative embodiments, the protein sequence, as provided herein, is processed in specific organelles, and this may require the addition of one or more localization signals, such as periplasmic localization sequences.

[0051] In alternative implementations, a protein-coding DNA sequence (e.g., as an expression vector) is inserted into the genome (stable or unstable), or alternatively, it can be additive. The recombinant protein expression system can be transient or permanent.

[0052] In alternative implementations, for example, to enhance the ability of a given protein to act as or as an antigen or immunogen for immunization purposes, the recombinant protein is purified; for example, the presence of impurities may cause immunized animals to produce antibodies against irrelevant targets; and the presence of excessive impurities may counteract the formation of a large amount of desired antibodies, and removing reactivity to impurities from polyclonal antibodies may be time-consuming and expensive.

[0053] In alternative embodiments, the purification of protein substances is based on the specific characteristics of the desired protein. For example, purification includes the use of hydrophobicity, charge and / or size, and chromatographic techniques such as hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), and / or size exclusion chromatography (SEC). In alternative embodiments, specific protein interactions are used for purification purposes, for example, using affinity purification, or using the lack of protein-specific properties to remove other protein substances, for example, using adsorption purification. In alternative embodiments, antibodies or other protein-specific binding proteins are used for affinity purification and / or adsorption purification of the protein.

[0054] In alternative embodiments, when recombinantly expressing a protein, a protein sequence that allows for specific purification methods is added, such as: epitope tags, such as FLAG, hemagglutinin (HA), c-myc, T7, Glu-Glu, ALFA tags, V5 tags, Myc tags, HA tags, Spot tags, T7 tags, and NE tags; biotin and streptavidin or avidin systems; multihistidine affinity tags, such as small HIS tags (6-8 amino acids) (and optionally immobilized metal affinity chromatography); an N-terminal glutathione S-transferase (GST) molecule followed by a protease cleavage site; a 43 kDa maltose-binding protein (MBP); an inteptide-chitin-binding domain (inteptide-CBD) tag; or a calmodulin-binding peptide (CBP) purification system that uses a C-terminal fragment from myosin light chain kinase to purify the protein of interest from bacteria. This increases the available tools for purification purposes and allows standard methods to be used for many different proteins.

[0055] In some cases, it is necessary to remove such purified sequences before performing immunization. This can be achieved by placing a protease site between the purified sequence and the actual protein-coding sequence, such as a chimeric protein sequence as presented herein. For example, the tobacco etch virus (TEV) protease leaves only an N-terminal glycine residue after cleaving the shared sequence.

[0056] In alternative embodiments, the recombinant proteins provided herein are produced in situ in an immunized animal, for example, by modifying cells in the animal to have novel or altered DNA sequences capable of encoding the expression of the recombinant proteins and expressing and / or secreting these immunogenic proteins.

[0057] Immunization process In alternative embodiments, methods are provided for producing antibodies or generating or stimulating immune responses in animals, such as mammals (e.g., rabbits, rodent species such as mice or rats, sheep, goats, pigs, cattle, or horses) or pheasant species (e.g., chickens), said methods comprising administering chimeric or recombinant proteins as provided herein.

[0058] In an alternative implementation, to obtain a polyclonal antibody against a protein target, an immune response is generated in another type of animal (species) using a protein derived from one type of animal (species).

[0059] In alternative embodiments, chimeric or recombinant proteins comprising at least one human epitope, as provided herein, are used to stimulate the immune system, for example, to generate a humoral immune response in mice, rats, rabbits, sheep, goats, pigs, cattle, horses, or chickens, and one or more derived or generated polyclonal antibodies can specifically recognize human proteins and can be used to specifically recognize, label, bind, and / or isolate human proteins from which at least one human epitope is derived.

[0060] In an alternative implementation, proteins from any species can be used to immunize another species to generate a humoral immune system, provided that the protein used for immunization carries at least one modification (e.g., at least one amino acid difference) compared to any homologous protein or protein domain in the species being immunized.

[0061] In alternative embodiments, an adjuvant is also used when administering chimeric or recombinant proteins as provided herein. While the administered chimeric or recombinant protein is a reagent that guides the immune response to produce antibodies against specific epitopes expressed by the recombinant protein, the adjuvant mixed with the protein ensures that the immune system is activated; for example, by using the adjuvant, the protein can be placed in a deposit that is released into the body over a longer period of time. In alternative embodiments, different adjuvants are used, for example, adjuvants based on various principles (such as the oil-in-water principle), such as Freund's adjuvant. In alternative embodiments, the protein and adjuvant mixture is injected into one or more subcutaneous sites. In alternative embodiments, the administration process is repeated several times (e.g., between about 2 and 10 times) to enhance the immune response (enhancement phase); and, high yields of polyclonal antibodies can be maintained by re-immunizing periodically but typically at longer intervals (e.g., every 3 to 16 weeks).

[0062] Select the epitopes to be grafted onto the protein backbone. In an alternative embodiment, a recombinant polypeptide is provided comprising a portion of a first polypeptide from a first species and at least one portion of a second polypeptide from a second species, wherein the at least one portion of the second polypeptide is a homolog of the first polypeptide, and wherein the homologous portion of the second polypeptide contains an epitope not present in the first polypeptide. In an alternative embodiment, homologous proteins or protein domains are present in both species of interest.

[0063] In an alternative embodiment, homologous proteins are proteins with similar three-dimensional structures; when proteins have more than 30% sequence similarity, they have the same 3D structure in 90% of cases, and proteins with much lower sequence identity may also have similar 3D structures. In an alternative embodiment, the 3D structural similarity between proteins is assessed using a distance matrix alignment (DALI) method, and empirically, a Z score higher than 8 indicates homology, while scores from 2 to 8 represent gray areas.

[0064] In an alternative implementation, the cytoskeleton protein or the first polypeptide from the first species is derived from the species to be immunized (species one), while the epitope sequence is derived from the species to be recognized by the polyclonal antibody (species two).

[0065] In an alternative implementation, the epitope sequence to be inserted into or constructed into the "background" protein, or the first polypeptide from the first species, is obtained by: First, the two amino acid sequences are compared, and differences of even a single amino acid residue are highlighted. Choose at least one (or more) such amino acid residue differences; and The backbone sequence (species 1) is modified by changing selected amino acids or multiple selected amino acids.

[0066] In an alternative embodiment, after introducing one or more selected epitopes into the backbone sequence, the derived heterozygous (or chimeric) protein is recombinantly expressed and optionally purified for use in immunization of species one, and the resulting polyclonal antibody (or monoclonal antibody derived from such humoral reaction) can be used to recognize the protein in species two.

[0067] In an alternative embodiment, the mixed or chimeric protein is considered ready for immunization if it can be maintained in solution at a concentration of at least about 50 µg / mL for at least one day. Further quality control may optionally be performed prior to immunization via immunological and / or biochemical tests or by spectroscopic examination (e.g., circular dichroism) to confirm the correct protein structure.

[0068] In alternative embodiments, when the polyclonal antibody is to be used for assays of intact proteins, such as ELISA, turbidimetry, and CLIA assays, including additional steps may be beneficial, for example: - Compare two amino acid sequences and highlight differences, even if there is only one amino acid residue. - These differences are highlighted in the 3D structure of the protein or domain; - Identify differences in residing in exposed areas of the surface; - Select at least one such surface exposure difference; and / or, - The backbone sequence (species one) is modified by changing selected amino acids to those of species two.

[0069] In some cases, the 3D structure of a protein or domain may be unknown and the second and third steps cannot be applied; instead, in alternative implementations, a range of heterozygous proteins with different epitope sequences are examined until the desired antibody is obtained.

[0070] Examples of the preparation and use of chimeric proteins as provided in this article Preventing undesirable antibody reactivity or properties In alternative embodiments, the recombinant polypeptides provided herein are used for the following, or the methods provided herein further include the following: - To improve the specificity for a homologous protein species in the family, for example by avoiding the application of an epitope from the backbone sequence of species II that is present in other members of the protein family (in species II), so that the immune response will target or be more focused on the remaining epitopes that are more unique to the selected protein species. - To increase the specificity of one of the multiple domains in a protein; this can be done by removing epitopes present in other domains of the protein family (of species two), so that the immune response will target the remaining epitopes that are more specific to the selected domain. - To enhance the synergistic effect of polyclonal antibody compositions for a given application; one example is to obtain reactivity against a subset of epitopes to induce rapid and efficient cross-binding in turbidimetric reactions; and another example is to create polyclonal antibodies that are capable of synergizing with monoclonal antibodies in assays such as ELISA or CLIA (e.g., by removing epitopes recognized by monoclonal antibodies). - To prevent undesirable properties of polyclonal antibodies, for example, by selectively removing epitopes from the species-two sequence, thereby avoiding complementary site isotypes on the antibody, where key properties such as antibody isoelectric point (pI) and hydrophobicity can be influenced or controlled to obtain desired properties when interacting with other materials (e.g., with plastic surfaces). - Gain greater control over the polyclonal antibody manufacturing process, making it more standardized between batches; one example is removing one or more immunodominant epitopes from the species-two sequence until a more consistent reactivity to minor epitopes is achieved in immunized animals; another example is eliminating or removing the weakest epitopes from the species-two polypeptide to avoid more variable responses to such epitopes; and / or - Reduce reactivity (e.g., reaction rate) to a given protein by removing some epitopes and / or reducing antibody affinity by using one or more modified epitopes (or inserting one or more modified epitopes into a species-2 sequence), where this can be used in applications such as wide-area turbidimetric assays.

[0071] Add the desired reactivity or properties In alternative embodiments, the recombinant polypeptides provided herein are used for the following, or the methods provided herein further include the following: - Multi-species reactivity allows the same antibody to be used for diagnosis in both human and animal species, for example; such antibodies can be made by inserting additional epitopes into a species-based backbone, or by combining or fusing different recombinant proteins with different epitope properties or different newly inserted epitopes. - Multi-protein reactivity, which enables polyclonal antibodies to recognize all or selected subsets of a protein family; this can be achieved by adding or inserting different epitopes between family members into a species-backbone, or by combining heterozygous proteins with different versions of selected epitopes in an immune mixture. - Multidomain reactivity enables polyclonal antibodies to recognize all or a selected subset of domain types; this can be achieved by adding different epitopes between domains to a species-based backbone, or by combining hybrid domains with different versions of selected epitopes in an immune mixture. - Reactivity to epitopes that fail to elicit a primary response; the lack of a primary response to a given epitope can be overcome by using a series of modified epitopes, as demonstrated, for example, by Escolano et al., 2016, Cell 166, 1445–1458, in the development of antiviral vaccines; this method of using sequential immunization can also be used to produce polyclonal antibodies; and / or - Enhance the desired properties of polyclonal antibodies, for example, by selectively removing epitopes from species-two sequences to avoid complementary site isotypes on the antibody; key properties, such as the antibody's pI and hydrophobicity, can be influenced or controlled to obtain the desired properties when interacting with other materials, such as when interacting with plastic surfaces.

[0072] In an alternative implementation, humoral immunity is an immune response involving the transformation of B cells into plasma cells, which produce and secrete antibodies against specific antigens.

[0073] In an alternative implementation, an epitope (also known as an antigenic determinant) is a portion of an antigen that is recognized by an antibody.

[0074] In an alternative implementation, the complementary site (also known as the antigen-binding site) is the part of the antibody that recognizes and binds to the antigen.

[0075] In an alternative implementation, the isoelectric point (pI) is the pH of the solution when the net charge of the protein becomes zero; when the solution pH is higher than pI, the surface of the protein is mainly negatively charged, and therefore molecules with the same charge will exhibit repulsive forces.

[0076] Vaccines and vaccination In alternative embodiments, vaccine formulations are provided comprising chimeric or recombinant polypeptides, nucleic acids encoding them (including molecules encoding DNA and RNA proteins (e.g., mRNA encoding proteins)) or nucleic acid expression vectors as provided herein, and / or cells as provided herein.

[0077] In alternative embodiments, vaccine formulations as provided herein may contain or further contain adjuvants or incomplete adjuvants, or pharmaceutically acceptable excipients, wherein optionally pharmaceutically acceptable excipients include sterile buffers, physiological saline, or water.

[0078] In alternative embodiments, chimeric or recombinant polypeptides, nucleic acids encoding them (such as RNA encoding proteins), or nucleic acid expression vectors as provided herein are formulated into liposomes, for example, as liposome delivery carriers having polycationic lipid compositions (e.g., cationic liposomes) and / or liposomes having a cholesterol backbone conjugated with polyethylene glycol, wherein exemplary cationic liposome compositions comprise or are made using the following substances: N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and cholesterol, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) and cholesterol, 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)-imidazoline chloride (DOTIM) and cholesterol, dimethyloctadecylammonium bromide (DDAB) and cholesterol, and combinations thereof.

[0079] For example, in an alternative embodiment, the nucleic acid encoding the protein may be DNA encoding one or more immunogenic peptides or proteins, and said DNA may be carried in an expression vector, such as a viral vector, for example an adenovirus vector, such as Ad5 or an adeno-associated virus vector (AAV). In an alternative embodiment, the recombinant adenovirus used in the vaccines provided herein may be as described in U.S. Patent Application No. 20200399323 A1, which describes, for example, a recombinant adenovirus containing a deletion in the E1 region or any deletion that results in viral replication defects, for example, the replication-defective virus may contain deletions of one or more of the E1, E3, and / or E4 regions; or, as described in U.S. Patent Application No. 20190382793 A1, which describes how to prepare a recombinant adenovirus for gene therapy.

[0080] In an alternative embodiment, the nucleic acid encoding the protein may be RNA, such as mRNA, which may be formulated into a lipid formulation or liposome and injected, for example, intramuscularly (IM), using a formulation and method as described in U.S. Patent Application No. 20210046173 A1, which describes the delivery (e.g., via intramuscular injection) of an immunogenic composition to a subject comprising RNA (e.g., mRNA) containing an open reading frame (ORF) containing an immunogenic or antigenic sequence as provided herein (or consisting of, or substantially consisting of, the immunogenic or antigenic sequences as provided herein); wherein optionally, the RNA (or an expression vector carrying DNA) is formulated into liposomes or lipid nanoparticles. LNP) or nanoliposomes, said liposomes, or lipid nanoparticles or nanoliposomes comprising: a non-cationic lipid comprising a mixture of cholesterol and DSPC, or a PEG-lipid, or a PEG-modified lipid, or an LNP, or an ionizable cationic lipid; or a mixture of (13Z,16Z)-N,N-dimethyl-2-nonyltetradecano-12,15-diene-1-amine, cholesterol, DSPC and PEG-2000 DMG. In alternative embodiments, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dispalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), or the PEG-lipid is PEG (PEG-DMG) coupled with dimyristoylglycerol.

[0081] In alternative embodiments, the chimeric or recombinant peptides, the nucleic acids encoding them, or the nucleic acid expression vectors provided herein are formulated with or administered with an adjuvant, which may include, for example, aluminum hydroxide or mineral oil; immunostimulants such as lipid A; Bordetella pertussis (…). Bortadella pertussis ) or Mycobacterium tuberculosis ( Mycobacterium tuberculosisProteins derived from these sources; for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Rahway, NJ); AS-2 (GlaxoSmithKline, Philadelphia, Pa.); aluminum salts, such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derived polysaccharides; polyphosphazenes; biodegradable microspheres; monophospholipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, interleukin-7, interleukin-12, and other similar growth factors, may also be used as adjuvants.

[0082] In alternative embodiments, chimeric or recombinant peptides, the nucleic acids encoding them, or nucleic acid expression vectors, as provided herein, may be administered in one or more dosage regimens.

[0083] In alternative embodiments, chimeric or recombinant peptides, nucleic acids encoding them, or nucleic acid expression vectors, as provided herein, or vaccines provided herein, may be administered at a dose between about 100 µg and about 1 mg; or at a dose comprising between about 50 µg and about 500 µg; or at a dose between about 1 mg and about 10 mg. The vaccine may be administered, for example, as a single dose, or as two, three, four, or five or more doses. In one embodiment, two doses are administered at a one-week or two-week interval.

[0084] In alternative embodiments, chimeric or recombinant peptides, nucleic acids encoding them, nucleic acid expression vectors, or vaccines, as provided herein, may be administered via intradermal, transdermal, intranasal (e.g., via intranasal drops or intranasal aerosol), intramuscular, subcutaneous, or sublingual routes.

[0085] In alternative embodiments, chimeric or recombinant peptides, nucleic acids encoding them, nucleic acid expression vectors, or vaccines, as provided herein, are administered using a syringe, pneumatic syringe, or jet injection device.

[0086] Manufacturing products and reagent kits Manufacturing products and kits are provided for practicing the methods provided herein, the manufacturing products and kits comprising, for example, nucleic acids, such as expression vectors for expressing chimeric or recombinant peptides as provided herein; or chimeric peptides as provided herein; or cells expressing chimeric or recombinant peptides as provided herein; or vaccine formulations as provided herein, for example, vaccine formulations comprising chimeric or recombinant peptides as provided herein; and optionally, the manufacturing products and kits may further include instructions for use for practicing the methods provided herein.

[0087] Any of the foregoing aspects and implementations may be combined with any other aspects or implementations disclosed herein in the Summary of the Invention, the Drawings and / or the Detailed Description sections.

[0088] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural referents.

[0089] Unless otherwise specified or obvious from the context, as used herein, the term “or” is understood to be inclusive and encompasses both “or” and “and”.

[0090] Unless otherwise specified or obvious from the context, as used herein, the term “about” is understood to mean within the normal tolerance range in the field, such as within 2 standard deviations of the mean. “About” (the use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. All numerical values ​​provided herein are modified by the term “about” unless clearly stated otherwise from the context.

[0091] Unless specifically stated or obvious from the context, as used herein, the terms “substantially all,” “substantially most,” “substantially all,” or “most” cover at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of the reference amount of the composition.

[0092] The entire contents of each patent, patent application, publication, and document cited herein are incorporated herein by reference. Reference to any of the aforementioned patents, patent applications, publications, and documents does not constitute an admission that any of them is applicable prior art, nor does it constitute any admission of the content or dates of such publications or documents. Individual citation of these documents should not be construed as an assertion or admission that any portion of any document is considered necessary material to satisfy the statutory disclosure requirements of any national or regional patent application. Nevertheless, the right to rely on any such documents where appropriate to provide material that an examining authority or court deems essential to the claimed subject matter is reserved.

[0093] Modifications to the foregoing can be made without departing from the basic aspects of the invention. Although the invention has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes can be made to the embodiments specifically disclosed herein, and such modifications and improvements remain within the scope and spirit of the invention. The invention described herein can be suitably practiced in the absence of any elements not specifically disclosed herein. Therefore, for example, in each instance herein, any of the terms “comprising,” “substantially consisting of,” and “consisting of” can be replaced by any of the other two terms. Thus, the terms and expressions used are used as descriptive rather than limiting terms, and equivalents of the features shown and described or portions thereof are not excluded, and it should be recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0094] The invention will be further described with reference to the embodiments described herein; however, it should be understood that the invention is not limited to such embodiments.

[0095] Example Unless otherwise stated in the examples, all recombinant DNA techniques were performed according to standard protocols, such as those described in Sambrook et al., (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY, and in Ausubel et al., (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references to standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, NY; and Brown (1998) Molecular Biology LabFax, 2nd Edition, Academic Press (UK), Volumes 1 and 2. Standard materials and methods for polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press and McPherson et al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0096] Example 1: Illustrative Method Materials and methods Epitope selection Human epitopes were identified based on sequence alignment. Chimeric variants were designed using the identified epitopes, which react with the constant structural domain of the λ-free light chain in humans, rather than rabbits.

[0097] Protein expression and purification The chimeric variant encoding λ-FLC was ordered from GenScript® and cloned into the expression pET22b(+) for E. coli expression. A ferritin-fused CdV6 was ordered from GENEART and cloned into the pTT5 vector for HEK cell expression. The E. coli λ-FLC construct was designed with an N-terminal His tag followed by a TEV cleavage site to separate the His tag from the constant domain. The recombinant rabbit λ-FLC constant domain grafted with all or a single epitope was expressed in the periplasm using E. coli strain BL21 (Invitrogen™). The ferritin-fused CdV6 construct was designed to be His-tag-free.

[0098] When expressing the recombinant protein in *E. coli*, lysogenic broth containing the recombinant protein was dialyzed against binding buffer (20 mM Na₂HPO₄, 150 mM NaCl, pH 7.4), and then the recombinant protein was immobilized by affinity chromatography (IMAC) using a His-tagged column (Cytivia). The immobilized protein was washed with wash buffer (20 mM Na₂HPO₄, 1 M NaCl, 20 mM imidazole, pH 7.4) at least 15 column volumes and eluted with elution buffer (20 mM Na₂HPO₄, 150 mM NaCl, 500 mM imidazole, pH 7.4). The His-tagged recombinant protein was dialyzed against cleavage buffer (20 mM Tris, 150 mM NaCl, pH 8), and then TEV protease (0.2 mg / mL, final concentration) supplemented with 2 mM reduced L-glutathione was added. The cleavage reaction was carried out overnight at +4°C. To separate the cleaved recombinant protein from the His tag, TEV protease, and uncleaved recombinant protein, the mixture was loaded onto a His-tagged column, and the eluent containing unlabeled recombinant protein was collected. The sample was further purified using size exclusion chromatography (SEC) Superdex 75™ Prep Grad (GE Healthcare) with binding buffer as the elution medium.

[0099] When expressed in HEK cells, a standard protocol was followed. The secreted protein was purified by concentrating the supernatant and loading it directly onto an S400 sephacryl (Cytiva) column. This process was repeated until high purity was achieved.

[0100] Protein concentration determination Protein concentration was calculated using A280 absorbance and sequence-specific elongation coefficients calculated using ProtParam (Expasy.org).

[0101] SDS-PAGE and Western blotting Protein purity was tracked using SDS-PAGE with pre-prepared NuPage™ 4-12% Bis-Tris gel (Invitrogen™). All protein samples were loaded with SDS sample buffer (350 mM Tris.HCl, 357 mM sodium dodecyl sulfate, 44.6% glycerol, 179 µM bromophenol blue, pH 6.8) and run on MES SDS running buffer (Novex). ® The procedure was performed using SimplyBlue™ (Invitrogen™). The gel was stained with SimplyBlue™. For Western blotting, NuPage™ 4-12% Bis-Tris gel (Invitrogen™) and MES SDS running buffer (Novex) were used. ® Proteins were isolated using [method name missing]. Electroblotting was performed at 30 V for 1 hour, and the proteins were transferred to a PVDF membrane (BioRad) in protein blotting buffer (25 mM Tris, 0.192 M glycine, and 25.3% ethanol). Following blotting, a blocking step was performed using blocking buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.5% Tween 20, pH 9.0). The blocked PVDF membrane containing the transferred proteins was incubated with primary antibody pAb (rabbit anti-E. coli diluted 1000-fold) at +4 °C with shaking for at least 1 hour and overnight. Before incubating the membrane with secondary antibody pAb (porcine anti-rabbit) for 1 hour, the membrane was washed with blocking buffer 4 × 10⁻⁶ min. After incubation with secondary antibody pAb, the membrane was washed again with blocking buffer 4 × 10⁻⁶ min and incubated with DAB (diaminobenzidine) and substrate for 20 min.

[0102] Antigen preparation and immunization High-purity antigen samples were produced based on SDS-PAGE and Western blot analysis of fractions purified from SEC. The antigen samples consisted of equal volumes of five chimeras, each carrying a single epitope (EP1, Ep2, Ep3, Ep4, or Ep5), or a single chimeric domain (CdV6) carrying all epitopes, or CdV6-Ferrtin, to elicit an immune response. Immediately prior to subcutaneous immunization of three-month-old rabbits, the antigen samples were mixed with equal volumes (1:1) of Freunds incomplete adjuvant (FIA). Serum was collected every two weeks before immunization and after the final booster immunization. Serum was preserved by adding sodium azide (NaN3) to a final concentration of 15 mM and storing at 4°C.

[0103] Enzyme-linked immunosorbent assay (ELISA) All antigens, i.e., chimeric variants of λ-FLC, were diluted to 1 µg / mL with coating buffer (10 mM Na₂HPO₄, 145 mM NaCl, 0.1% Tween-20, pH 7.2). The dilutions were used to coat 96-well plates overnight at 4°C. All primary antibody pAbs (IgG fractions or antiserum) were serially diluted 3-fold from 100 µg / mL using 5% skim milk, or serially diluted 200-fold. Plates were washed with wash buffer (10 mM Na₂HPO₄, 500 mM NaCl, 0.1% Tween-20, pH 7.2) and incubated with the primary antibody pAbs at room temperature for 1 hour with agitation. The plates were then washed with wash buffer and incubated with secondary antibody pAbs (HRP goat anti-rabbit) diluted to 10 µg / mL in 5% skim milk for 1 hour with agitation. Finally, the plate was washed with washing buffer and developed for 5 minutes after adding 100 µL of TMB (DAKO S1599) to each well. The reaction was stopped by adding 100 µL of 0.5 M H2SO4 to each reaction well. The results were detected using an ELISA reader, SoftMax™ 6.2.1, at detection wavelengths of 450 nm and 650 nm.

[0104] This embodiment illustrates exemplary schemes and methods as provided herein.

[0105] In alternative embodiments, the methods provided herein utilize recombinant chimeric antigens. In alternative embodiments, constructs carrying a single epitope are prepared for immunization with a pool of single-epitaxy antigens.

[0106] Figure 1 An exemplary immunization method using an antigen with numerous human epitopes is illustrated. CDv6 is a rabbit λ free light chain (FLC) constant domain modified to carry epitopes at five positions; that is, CDV6 is a chimeric domain derived from the rabbit λ free light chain constant domain and grafted with human epitopes specific to the free light chain, and epitopes numbered 1 to 5 are single epitopes grafted onto the rabbit scaffold constant domain. CdV6 (SEQ ID NO:1) GQPAVTP T V T LFPPSSEELKDNKATLVCLI S DFYP GA V T VNWKADGNSVTQGV E TT K PSKQSNNKYAASS Y LSLSANQWKSYQSVTCQVTHEGHTVEKSLAP TECS (SEQ ID NO:1) The rabbit amino acid sequence from the constant domain of the (rabbit) λ free light chain is (SEQ ID NO:2): GQPAVTPSVILFPPSSEELKDNKATLVCLINDFYPRTVKVNWKADGNSVTQGVDTTQPSKQSNNKYAASSFLSLSANQWKSYQSVTCQVTHEGHTVEKSLAPAECS (SEQ ID NO: 2) The human amino acid sequence derived from the constant domain of the λ free light chain is (SEQ ID NO:3): GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:3) exist Figure 1 In this study, Ep1 through Ep5 antigens are similar to CDv6, but each antigen has only one human epitope: Epitope 1 (SEQ ID NO:4) GQPAVTP T V T LFPPSSEELKDNKATLVCLI S DFYPRTVKVNWKADGNSVTQGVDTTQPSKQSNNKYAASSFLSLSANQWKSYQSVTCQVTHEGHTVEKSLAPAECS Epitope 2 (SEQ ID NO:5) GQPAVTPSVILFPPSSEELKDNKATLVCLINDFYPRTVKVNWKADGNSVTQGV E TTQPSKQSNNKYAASS Y LSLSANQWKSYQSVTCQVTHEGHTVEKSLAPAECS Epitope 3 (SEQ ID NO:6) GQPAVTPSVILFPPSSEELKDNKATLVCLINDFYPRTVKVNWKADGNSVTQGVDTT K PSKQSNNKYAASSFLSLSANQWKSYQSVTCQVTHEGHTVEKSLAPAECS Epitope 4 (SEQ ID NO:7) GQPAVTPSVILFPPSSEELKDNKATLVCLINDFYPRTVKVNWKADGNSVTQGVDTTQPSKQSNNKYAASSFLSLSANQWKSYQSVTCQVTHEGHTVEKSLAP T ECS Epitope 5 (SEQ ID NO:8) GQPAVTPSVILFPPSSEELKDNKATLVCLINDFYP GA V T VNWKADGNSVTQGVDTTQPSKQSNNKYAASSFLSLSANQWKSYQSVTCQVTHEGHTVEKSLAPAECS In the case of CdV6-ferritin, CDV6 immunization was performed at 100 µg or 50 µg per rabbit, and immunization was performed using Ep1 to Ep5 single epitope chimeric antigens as a pool, with 20 µg of each chimera used. Therefore, in both cases, animals received 100 µg of antigen per immunization round. The immunization frequency and adjuvant formulation were identical in both groups.

[0107] These two approaches yielded different results, such as Figure 2 As shown, the figure graphically illustrates the results of immunization using CDv6 (blue circle) or pools Ep1 through Ep5 (orange squares). Polyclonal antibodies were compared using ELISA. 96-well plates were coated with Ep chimeric protein. The coated plates were then challenged serially with a primary antibody derived from immunized rabbits at three-fold dilutions. Secondary antibodies carrying HRP were used, with TMB as the substrate, and absorbance was reported. The structure of the constant domain with human epitopes is shown in the lower right corner. Each of the five titration plots represents the domain, with the associated epitope shown in a color corresponding to the epitope color in the lower right corner structure.

[0108] Immunization with CDv6 produced antibodies recognizing epitopes 1, 2, 4, and 5, but low antibody titers against epitope 3. In contrast, pools Ep1 through Ep5 gave high titers for all five epitopes.

[0109] Empirically, it's expected that some epitopes exhibit immunogenicity. They elicit a strong response in some way, and a large number of antibodies with low Kd (high affinity) are found. In this case, we see another reason for antibody response bias: one epitope is less effective than the other four and does not induce the required amount of antibody.

[0110] However, the same epitope effectively induces an antibody response when presented as a single epitope antigen, even when the other four epitopes are used simultaneously. This indicates that the epitope is fully immunologically active within the context of its protein domain.

[0111] Therefore, we anticipate that by using the methods presented herein, antibodies against all or most epitopes can be induced by immunizing pools of chimeric antigens, each carrying only a single epitope.

[0112] We also anticipate that, by using the methods provided herein, chimeric antigens carrying said epitopes can be prepared if several epitopes have similar intensities. Such multi-epitope antigens are advantageous because a smaller total amount of antigen can be used for each immunization.

[0113] We propose the term "epitope tuning" to describe the optimal combination of a single epitope and / or a selected combination of multiple epitopes carried by a chimeric antigen.

[0114] Some epitopes carry post-translationally modified (PTM) portions, such as thiol groups or phosphorylation sites. By using monoepisode constructs, specific modifications (e.g., dephosphorylation) can be applied, and immunization can be performed with all relevant versions. In this way, polyclonal antibodies exhibit the same specificity for all versions of PTM epitopes.

[0115] When it is important to obtain only polyclonal antibodies that recognize selected (human) epitopes, the epitopes are inserted into (rabbit) antigens or interspecies homologous antigenic domains, such as in the case of immunoglobulins in humans and rabbits.

[0116] If non-homologous or non-originating parts from the species to be immunized (rabbit) are used, these parts must be selected to not interfere with the (human) epitope. Furthermore, they will induce antibodies even when the ability of the selected epitope to induce antibodies is not impaired by such parts. It must be ensured that these non-epitope-recognizing antibodies do not interfere with the downstream application of polyclonal antibodies.

[0117] Antibodies are produced through mechanisms within an animal's adaptive immune response. Antigens are taken up and presented as small fragments in MHC proteins. Binding to immature immunoglobulins located on B cells leads to clonal expansion. Subsequent mutations and selection result in the production of high-affinity antibodies. These processes appear to be competitive, allowing some epitopes to induce large numbers of antibodies while simultaneously inhibiting those induced by other epitopes.

[0118] The construct of CdV6 (bold) fused to rabbit ferritin via a linker (italic) has also been used as an immunogen (the rabbit ferritin portion is underlined): CdV6- connector - Ferritin (SEQ ID NO:9) GQPAVTP T V T LFPPSSEELKDNKATLVCLI S DFYP GA V T VNWKADGNSVTQGV E TT K PSKQSNNKYAASS Y LSLSANQWKSYQSVTCQVTHEGHTVEKSLAP T ECS GGGGSGGGGSGGGGSGGGGSGGGGS MTSQIRQNYSPEVEA AVNHLVNLHLRASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREAAERLLKMQNQRGGRALFQDVQKPSQDEWGK TLNAMEAALALEKNLNQALLDLHALGSAHTDPHLCDFLENHFLDEEVKLLKKMGDHLTNIRRLSGPQASLGEYLFER LTLKHD Figures 3A to 3G The data presented demonstrates the superiority of using a single epitope construct for immunization: comparisons were made by ELISA of four different immunizations against five putative epitopes (Ep1, Ep2, Ep3, Ep4, Ep5, Ep6, Ep7, Ep8, Ep9, Ep10, Ep8, Ep9 ...9, Ep9, Ep9 Figure 3A ), Ep2 Figure 3B ), Ep3 Figure 3C ), Ep4 Figure 3C ) and Ep5 Figure 3E The reactivity of Cdv6: a rabbit λ light chain constant structural domain carrying all five human epitopes. Figure 3F CDV6+Oval: Cdv6 co-immunized with ovalbumin. Cdv6-Fer: Ferritin fused with Cdv6 and folded into a 24-mer structure. Epx+Oval: Five Cdv constructs, each carrying one of the human epitopes described above, mixed with ovalbumin. Wells were coated with a specified epitope-specific version of Cdv and incubated with antiserum from one of four different immunizations. HRP-labeled anti-rabbit antibodies were used for visualization purposes. Figure 3G The Cdv6 domain used as an antigen is schematically illustrated, with all five epitopes labeled 1 to 5 and highlighted with circles.

[0119] Table 6.

[0120] Table 6. Immunization using multiple single-epitope constructs provides significant responsiveness to all epitopes. EC 50 Value originates from Figures 3A to 3E Data from [source]. Calculate the average EC50 for each immunogen. 50 Value. *Note Ep5 EC 50 The value was estimated through curve fitting.

[0121] Example 2: Indicative method and construction of chimeric constructs This embodiment illustrates exemplary chimeric constructs, each carrying a human epitope in a rabbit context.

[0122] Myoglobin is an oxygen carrier in cardiac muscle tissue, composed of 154 amino acids and heme groups. It is a biomarker for heart disease; elevated serum concentrations serve as an early warning sign. Myoglobin is homologous between humans and rabbits, but differs by 16 amino acids (aa). Human myoglobin is predicted to contain six epitopes, such as... Figure 4 As indicated in the document.

[0123] Six chimeric constructs were generated, each encoding a rabbit myoglobin containing one of six human epitopes, and encoded antigens were obtained as described herein. Rabbits were immunized with pools containing each of the six antigens encoded by the constructs. Balanced immune responses against each of the six human epitopes in the antigens were obtained. No significant immune response was observed against the rabbit sequences in the antigens.

[0124] In contrast, when rabbits were immunized with antigens containing three to five human epitopes introduced into rabbit myoglobin, a more diverse response was observed, with antibodies targeting some human epitopes more often than others. Immunization with the chimeric construct did not elicit a significant immune response to native rabbit myoglobin.

[0125] Rabbit myoglobin (see Figure 4 ) (SEQ ID NO:10) MGLSDAEWQLVLNVWGKVEADLAGHGQEVLIRLFHTHPETLEKFDKFKHLKSEDEMKASEDLKKHGNTVLTALGAILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISEAIIHVLHSKHPGDFGADAQAAMSKALELFRNDIAAQYKELGFQG (SEQ ID NO:10) Human myoglobin ( Figure 4 The text also indicates the location of the bolded tabletops; see also... Figure 3G (circle in the image) (SEQ ID NO:11) MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG (SEQ ID NO:11) Example 3: An exemplary method for generating more diverse polyclonal antibodies We present a method for obtaining strong and stable polyclonal antibody responses. At the heart of this method is immunization using a mixture of chimeric constructs, each presenting only one human epitope, although in some cases the number of chimeric constructs can be reduced by adding multiple epitopes to each chimera.

[0126] Example 1 above relates to a constant domain from human λ-free light chain (λ-FLC). This is a protein domain tightly packed with so-called β-sheet folds. Another example is myoglobin. This protein is composed of α-helical elements and has a highly differentiated amino acid sequence.

[0127] In Example 1 above, the epitopes were located in a hidden surface and thus covered by intact IgG. Reactivity against all epitopes was observed by immunization with a constant domain grafted with all epitopes (CdV6, gray triangle, Fig. 3). However, epitope 3 showed significantly lower recognition compared to other epitopes (Ep1, Ep2, Ep4, and Ep5). The relatively low immunogenicity of this single domain is evident from the number of rabbits that responded to the immunization. Strong immune responses were obtained in all rabbits by co-immunizing CdV6 with oval protein. However, as can be seen from Fig. 3, the diversity of polyclonal antibodies was not improved (CdV6+Oval shape, blue circle).

[0128] Data indicate that one or more epitopes are dominant and sufficient to clear the immunogen in rabbits. Therefore, cloning is not performed targeting weaker epitopes. To test this hypothesis, we used rabbit boxes grafted with epitopes Ep1, Ep2, Ep3, Ep4, or Ep5, and immunized them with five chimeric proteins. Figure 3F The epitope is shown on the 3D structure in the image.

[0129] However, it should be emphasized that we used five different proteins, each carrying only one human epitope, such as... Figure 1 and Figure 5As shown in Figure 3, a high antibody response against Ep3 was also observed when a mixture of the five chimeric constructs was applied.

[0130] In the second embodiment using myoglobin described below, all epitopes are exposed to the solvent, but some epitopes may be inhibited due to steric hindrance (Figure 18). As described in Example 1 above, immunization with a pool avoids any steric hindrance.

[0131] Two groups of rabbits were immunized with either a pool of chimeras containing a single epitope (Epx) or human native myoglobin (hMyo) carrying all epitopes, such as... Figure 5 As shown. To stimulate the immune response, both groups were co-immunized with ovalbumin.

[0132] Materials and methods Epitope selection Human epitopes were identified based on sequence alignment. Chimeric variants were designed using these identified epitopes, which could induce antibody production against human myoglobin instead of rabbit myoglobin.

[0133] Protein expression and purification All chimeric variants encoding myoglobin were ordered from GenScript® and cloned into pET3c (Novagen) for expression. The myoglobin constructs were designed to be tagless. Recombinant myoglobin was expressed according to (Bianchi, M. et al., Protein Expr Purif. 2003 Jun; 29(2):265-71). In short, bacteria can biosynthesize heme by adding the heme precursor 5-aminolevulinic acid (ALA) to lysogenic broth (LB) medium, which allows myoglobin to be expressed in its full form. Full myoglobin was expressed in the cytoplasm for 20 hours. Cells were sonicated and centrifuged to precipitate cell debris before protein purification. The supernatant was dialyzed against binding buffer (20 mM Tris pH 7.4) and immobilized on an anion exchange column (Cytivia). After washing with binding buffer, myoglobin was eluted with sodium chloride solution of gradually increasing concentration. The eluted sample was further purified using SEC (Superdex 75™ Prep Grad [GE Healthcare]) with running buffer (20 mM Na2HPO4, 150 mM NaCl, pH 7.4).

[0134] Protein concentration determination Protein concentration was calculated using A280 absorbance and sequence-specific elongation coefficients calculated using ProtParam (Expasy.org). UV-Vis spectra were recorded to verify whole myoglobin as indicated by the Solvay bands.

[0135] SDS-PAGE and Western blotting Protein purity was tracked using SDS-PAGE with pre-prepared NuPage™ 4-12% Bis-Tris gel (Invitrogen™). All protein samples were loaded with SDS sample buffer (350 mM Tris.HCl, 357 mM sodium dodecyl sulfate, 44.6% glycerol, 179 µM bromophenol blue, pH 6.8) and run on MES SDS running buffer (Novex). ® The procedure was performed using SimplyBlue™ (Invitrogen™). The gel was stained with SimplyBlue™. For Western blotting, NuPage™ 4-12% Bis-Tris gel (Invitrogen™) and MES SDS running buffer (Novex) were used. ® Proteins were isolated using [method name missing]. Electroblotting was performed at 30 V for 1 hour, and the proteins were transferred to a PVDF membrane (BioRad) in protein blotting buffer (25 mM Tris, 0.192 M glycine, and 25.3% ethanol). Following blotting, a blocking step was performed using blocking buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.5% Tween 20, pH 9.0). The blocked PVDF membrane containing the transferred proteins was incubated with primary antibody pAb (rabbit anti-E. coli diluted 1000-fold) at +4 °C with shaking for at least 1 hour and overnight. Next, the membrane was washed with blocking buffer 4 × 10 min and then incubated with secondary antibody pAb (porcine anti-rabbit) for 1 hour. After incubation with secondary antibody pAb, the membrane was washed again with blocking buffer 4 × 10 min and incubated with DAB (diaminobenzidine) and substrate for 20 min.

[0136] Antigen preparation and immunization High-purity antigen samples were produced based on SDS-PAGE and Western blot analysis of fractions purified from SEC. The final antigen samples contained only fractions free of any visible impurities. The antigen samples contained equal amounts of five chimeric domains, each carrying a single epitope (EP1, Ep2, Ep3, Ep4, or Ep5); or a single domain carrying all epitopes (native human myoglobin [Lee Bioscience]) to elicit an immune response. Before subcutaneous immunization of rabbits, the antigen samples were mixed with an equal volume (1:1) of Freund's incomplete adjuvant (FIA). Serum was collected every two weeks before immunization and after the final booster immunization. Serum was preserved by adding sodium azide (NaN3) to a final concentration of 15 mM and storing at 4°C.

[0137] Enzyme-linked immunosorbent assay (ELISA) All antigens prepared from chimeric variants of myoglobin were diluted to 3 µg / mL. The diluent was used to coat 96-well plates overnight at 4°C. All primary antibody pAbs (antiserum) were serially diluted 3-fold starting at 100 µg / mL with 5% skim milk, or 200-fold. Plates were washed with wash buffer (10 mM Na₂HPO₄, 500 mM NaCl, 0.1% Tween-20, pH 7.2) and incubated with the primary antibody pAb at room temperature for 1 hour with agitation. Subsequently, the plates were washed with wash buffer and incubated with secondary antibody pAb (HRP goat anti-rabbit) diluted to 10 µg / mL in 5% skim milk for 1 hour with agitation. Finally, the plates were washed with wash buffer and developed for 5 minutes after adding 100 µL of TMB (DAKO S1599) to each well. The reaction was stopped by adding 100 µL of 0.5 M H₂SO₄ to each well. Results were detected using an ELISA reader with SoftMax™ 6.2.1 at detection wavelengths of 450 nm and 650 nm.

[0138] Table 1 (for Example 3), for Figure 7 Summary, discussion and conclusions regarding myoglobin results Results Summary Animals immunized with epitope mixture (Epx) had higher antibody titers against human myoglobin compared to animals immunized with human myoglobin. Figure 10 and Figure 11In ELISA assays, superior induction of antibody production was evident when human myoglobin and any of the five chimeric constructs were used as decoys. This demonstrates that the chimeric immune principle is effective for myoglobin as well as for the structurally and sequence-dissimilar constant λ light chain domain.

[0139] We compared the titer differences between the first and second blood samples from each rabbit (Figures 12 and 14) and applied the EC50 value ( Figure 13 and Figure 15 Perform a paired t-test. Figure 16 This indicates that an additional round of Epx immunization increased the response to epitope 1 (p=0.03) and possibly to epitope 3 (p=0.06), but native human myoglobin, as well as epitopes 2, 4, and 5, did not show an increase in mean EC50 values. According to paired t-tests, immunization with native human myoglobin did not increase the overall level of antibodies against any of Ep1 through Ep5 or against human myoglobin.

[0140] exist Figure 17 In the diagram, the EC50 value of the first blood sample is plotted on the x-axis, and the EC50 ratio between the second and first blood samples is plotted on the y-axis. Only data from the Epx immunization group are shown. The inverse relationship is evident, indicating that a higher EC50 value predicts less titer increase (or even decrease).

[0141] Overall, if the Epx immunization program continues, the titer against Ep1 is likely to continue to rise, as the titer is low and has been shown to increase from the first blood draw to the second.

[0142] It is even more difficult to predict the titer results for Ep3. On the one hand, the average titer of the four rabbits may indeed have increased from the first blood collection to the second, but on the other hand, according to... Figure 17 A high titer makes it less likely to increase further.

[0143] Rabbit #3 is special because it has very high titers for Ep2, Ep4, and Ep5. Figure 15 The titer decreased from the first blood sample to the second. This extreme example may indicate an overshoot effect, where very high titer levels decrease over time. This is also interesting because it shows that chimeric immunization can induce very high titers against multiple epitopes in the same rabbit. Notably, while titers against epitopes 2, 4, and 5 decreased from the first to the second blood sample, titers against Ep1 and Ep3 increased (…). Figure 13 It appears that Rabbit 3 amplified the overall trend of the titers of the five epitopes stabilizing.

[0144] Figure 11 The results show that immunization with the epitope mixture (Epx) at the second blood draw yielded ELISA titers very similar to those for Ep2 through Ep5, but lower titers against Ep1. Meanwhile, our most certain prediction is that the titer against Ep1 will increase. Overall, this suggests that responses to these five epitopes will balance out over time.

[0145] discuss Animal immunization ultimately leads to the selection of plasma cells that produce antibodies, which are then stored in the bone marrow for future use. Each plasma cell expresses only one specific antibody sequence, and polyclonal antibodies are products expressed from multiple plasma cell clones.

[0146] However, this process sometimes exhibits the advantage of certain antibody clones, resulting in the titer of the polyclonal antibody being limited to epitopes on the target antigen to some extent.

[0147] We hypothesize that, for myoglobin, the overlap, particularly between epitopes 2, 3, and 5 (Figure 18), may be one reason for this limitation. If the B-cell clone recognizing epitope 3 is highly functional (e.g., if the antibody has high affinity and expression is efficient), the antigen may be bound and blocked before other B-cell clones bind to it. This would effectively prevent further induction of these clones.

[0148] At least one other mechanism should be considered: the uniqueness of the epitope. All else being equal, epitopes more likely to evolve highly specific antibodies are, in turn, more likely to induce high-titer antibodies. This leads to rapid antigen removal, which may hinder the evolution of alternative antibodies that bind to other epitopes. Similarly, this results in lower diversity of polyclonal antibodies.

[0149] Chimeric constructs enable effective uncoupling of the involved epitopes in immunotherapy, allowing the epitopes to function independently. Unique epitopes are still effectively targeted, but without negative outcomes for less attractive epitopes. It has been found that functional complementary sites of antibodies are over-occupied in antibody-antigen contact regions by aromatic residues (Tyr, Trp, and to a lesser extent Phe) (H. Peng, KH Lee, J. Jian, A. Yang, Origins of specificity and affinity in antibody–protein interactions, Proc. Natl. Acad. Sci. USA 111 (26) E2656-E2665, 2014). In the contact region, aromatic residues primarily interact with the skeletal atoms and side chains of the epitope through relatively weak non-covalent interactions. These weak interactions are the sum of face-to-edge or parallel π stacking, cationic or anionic π, hydrogen bond donors through aromatic π systems, alkyl carbons through CH-π interactions, and sulfur-aromatic interactions. Because all these interactions are relatively weak and are the sum of many interactions, a binding energy of kd ~ 1 nM is achieved. Aromatic residues are typically surrounded by hydrophilic residues (Ser, Thr, Asp, and Asn).

[0150] Epitopes are much more difficult to identify than complementary sites, where some patterns can be found. However, analysis of antibody-antigen interaction interfaces has revealed that hydrophobic interactions contribute very little to the driving forces in antibody-antigen recognition (H. Peng, KH Lee, J. Jian, A. Yang, Origins of specificity and affinity in antibody–protein interactions, Proc. Natl. Acad. Sci. USA 111 (26) E2656-E2665, 2014).

[0151] In the case of myoglobin, Ep3 induces a rapid, high-level response, while Ep1 induces a much slower evolutionary response. Interestingly, the Ep1 epitope exhibits characteristics of a weakly inducible epitope. Because Pro, Leu, and Ala are hydrophobic residues, they can only be classified as weakly immunogenic. Furthermore, Leu is incorporated into the core and therefore not exposed to the solvent, thus preventing side-chain interactions.

[0152] in conclusion We conclude that, during early immunization, immunizing rabbits with a mixture of recombinant myoglobin chimeras (each chimera carrying only one human epitope) increases antibody production against the selected epitopes more rapidly than when using human myoglobin as the antigen. The data support the superiority of immunization with epitope pools (Epx) over producing antibodies against human epitopes present in the mixture. Importantly, these data obtained early in immunization indicate that while Ep2 through Ep5 initially induce high antibody expression, this expression subsequently plateaus or even declines, while titers against Ep1 steadily increase, thereby stabilizing the polyclonal response.

[0153] Refer to Example 1 Angeletti et al., Outflanking immunodominance to target subdominant broadly neutralizing epitopes. 2019. PNAS, Vol. 116, No. 27, pp. 13474-13479. Immunology and Evolution of Infectious Disease. Chapter 6: Immunodominancewithin hosts. Steven A. Frank. Princeton University Press (2002).

[0154] Price et al., Light-scattering immunoassay of specific proteins: areview. (1983). Ann Clin Biochem 20, pp. 1-14.

[0155] Reference Example 3 Bianchi, M. et al., Protein Expr Purif. June 2003; 29(2):265-71 H. Peng, KH Lee, J. Jian, A. Yang, Origins of specificity and affinityin antibody–protein interactions, Proc.Natl.Acad.Sci.USA 111 (26) E2656-E2665, (2014) Several embodiments of the present invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, other embodiments are within the scope of the claims.

Claims

1. A method for generating a balanced immune response against multiple epitopes present in an antigen, the method comprising immunizing a host with multiple polypeptides, wherein each polypeptide comprises one epitope or a subset of the multiple epitopes present in the antigen.

2. The method for generating a balanced immune response according to claim 1, wherein the balanced polyclonal antibody response is a polyclonal antibody response comprising (or resulting in) multiple polyclonal antibody groups, each polyclonal antibody group specifically binding to a different epitope on the antigen, and none of the epitopes having a significant advantage over the others, thereby generating a balanced polyclonal antibody response or a balanced polyclonal serum.

3. The method for generating a balanced immune response according to claim 1, wherein the balanced polyclonal antibody response or balanced polyclonal serum comprises multiple antibody groups with substantially similar titers.

4. The method for generating a balanced immune response according to claim 2, wherein in the polyclonal response, none of the plurality of polyclonal antibody groups has about 5%, 10%, or 30% or more more antibodies than any other polyclonal antibody group in the plurality of polyclonal antibody groups.

5. The method for generating a balanced immune response according to claim 2, wherein in the polyclonal response, none of the plurality of polyclonal antibody groups has a signal intensity, signal-to-noise ratio, or signal rate that is approximately 5%, 10%, 20%, or 30% higher than any other polyclonal antibody group in the plurality of polyclonal antibody groups.

6. The method for generating a balanced immune response according to claim 2, wherein none of the epitopes is significantly superior to the others, meaning that each antibody group has a binding affinity, or affinity constant K, for its corresponding antigen. a and / or dissociation constant K d Compared to all other antibody groups, the difference is no more than about 5%, 10%, 20%, or 30%.

7. The method for generating a balanced immune response according to claim 1, the method further comprising identifying whether one or more dominant epitopes exist among the plurality of epitopes.

8. The method for generating a balanced immune response according to claim 7, wherein the one or more dominant epitopes are identified by: immunizing a first species with the antigen, identifying the polyclonal antibody group generated in the first species and the epitopes binding to the polyclonal antibody group, and determining whether one or more epitopes or polyclonal antibody groups are dominant compared to other epitopes or polyclonal antibody groups.

9. The method for generating a balanced immune response according to claim 7, wherein the first epitope is considered superior to the other epitopes if the antibody group binding to the first epitope has a titer at the first epitope that is at least about 5%, 10%, 20%, or 30% higher than the binding affinity of the other antibody groups to their respective epitopes.

10. The method for generating a balanced immune response according to claim 8, the method further comprising engineering the modified antigen by: removing one, several, or all of the identified one or more dominant epitopes from the antigen, or modifying the structure of the antigen responsible for creating the one or more dominant epitopes such that the one or more dominant epitopes are no longer immunogenic in the first species, or have significantly lower immunogenicity in the first species.

11. The method for generating a balanced immune response according to claim 10, wherein a significant reduction in immunogenicity means a reduction in immunogenicity of at least about 85%, 90%, or 95%.

12. The method for generating a balanced immune response according to claim 1, the method further comprising isolating or substantially purifying the polyclonal antibody of the immune host.

13. The method for generating a balanced immune response according to claim 1, wherein the antigen comprises a plurality of epitopes, including proteins, optionally recombinant chimeric proteins.

14. The method for generating a balanced immune response according to claim 13, wherein the recombinant chimeric antigen polypeptide comprising multiple epitopes comprises: (a) Polypeptides derived from the first species, and (b) at least one epitope comprising a heterologous amino acid sequence or amino acid residue derived from at least a second species. At least one heterologous amino acid sequence or amino acid residue derived from the second species is inserted into, linked to, created in, or replaces or substitutes the amino acid sequence of the polypeptide derived from the first species; Furthermore, the amino acid sequence of the recombinant chimeric antigen polypeptide is essentially composed of amino acid sequences derived from the first species. Furthermore, when the amino acid sequence from the second species is inserted into, linked to, created in, or replaces or substitutes for a portion of the amino acid sequence of the polypeptide from the first species, at least one new epitope is generated, formed, or created on the polypeptide from the first species. When the recombinant chimeric antigen polypeptide is applied to the first species, the new epitope enables the first species to generate a humoral antibody response specific to the at least one new epitope. When the recombinant chimeric antigen polypeptide is used to generate a humoral immune response from an animal of the first species, the polyclonal antibody generated in the first species binds substantially only specifically to the at least one novel epitope and substantially nonspecifically to a polypeptide derived from the first species that lacks the at least one novel epitope, or an epitope created, formed, or generated by the at least one heterologous amino acid sequence or amino acid residue derived from the second or additional species, wherein the at least one heterologous amino acid sequence or amino acid residue is inserted into, linked to, created in, or replaces or substitutes the portion of the polypeptide derived from the first species.

15. The method for generating a balanced immune response according to claim 14, wherein the recombinant chimeric antigen polypeptide comprising multiple epitopes derived from the second species is a homolog of the polypeptide derived from the first species.

16. The method for generating a balanced immune response according to claim 14, wherein the amino acid sequence from the at least one second species is homologous to the first species and is inserted into a portion of the amino acid sequence of the polypeptide derived from the first species, linked to the portion, created in the portion, or replaces or substitutes the portion or the at least one homologous second species sequence, replacing all or almost all structurally homologous segments or portions of the amino acid sequence of the polypeptide derived from the first species.

17. The method for generating a balanced immune response according to claim 14, wherein the amino acid sequence of the at least one second species is homologous to the first species, and the at least one homologous second species sequence that is inserted into, linked to, created in, or replaces the portion or substitutes for the portion is structurally homologous to the amino acid sequence of the polypeptide from the first species.

18. The method of claim 14, wherein the homolog of the first species has at least about 25% to 99% sequence identity with its homolog in the second species.

19. The method of claim 14, wherein the homolog of the first species has substantially the same secondary and / or tertiary structure as its homolog in the second species.

20. The method according to claims 18 and 19, wherein the homolog of the first species has at least about 25% to 99% sequence identity with its homolog in the second species and has substantially the same secondary and / or tertiary structure as its homolog in the second species.

21. The method of claim 14, wherein the homolog of the first species has at least about 50% sequence identity with its homolog in the second species.

22. The method of claim 21, wherein the homolog of the first species has at least about 70% sequence identity with its homolog in the second species.

23. The method of claim 12, wherein the homolog of the first species has at least about 80% sequence identity with its homolog in the second species.

24. The method of claim 12, wherein the homolog of the first species has at least about 90% sequence identity with its homolog in the second species.

25. The method according to any one of claims 14 to 26, wherein the amino acid sequence from the first species and the amino acid sequence from the second species have a Z score of about 2 to about 8 when aligned using a distance matrix alignment.

26. The method of claim 25, wherein the amino acid sequence from the first species and the amino acid sequence from the second species have a Z score of at least 8 when aligned using a distance matrix alignment.

27. The method according to any one of claims 14 to 26, wherein the polypeptide derived from the first species and its homologous polypeptide derived from the second species are antibodies.

28. The method according to any one of claims 14 to 27, wherein the polypeptide derived from the first species and the at least one heterologous amino acid sequence derived from the second species are both derived from the antibody heavy chain or the antibody light chain.

29. The method of claim 28, wherein the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain, or the light chain is a κ or λ light chain.

30. The method according to any one of claims 14 to 29, wherein the first species is a mammal species; the second animal is a mammal species; or, the first species is an animal of the order Galliformes or family Phasianidae, and the second species is a mammal species.

31. The method according to any one of claims 14 to 30, wherein the first species is a rabbit, a rodent species, a sheep, a goat, a pig, a cow, a horse, or a chicken; and the second species is a human.

32. The method of claim 31, wherein the rodent species is a rat or a mouse.

33. The method according to any one of claims 14 to 32, wherein at least about 80% to about 99% of the amino acid sequence of the recombinant chimeric antigen polypeptide is an amino acid sequence derived from the first species, and / or about 1% to about 20% of the amino acid sequence of the recombinant chimeric antigen polypeptide is an amino acid sequence derived from the at least one second species.

34. The method according to any one of claims 14 to 33, wherein one, two, three, four, five, six, seven, eight or more dominant epitopes are removed or deleted, or modified such that they are no longer immunogenic in the first species, or their immunogenicity in the first species is significantly reduced.

35. The method of any one of claims 14 to 34, wherein the recombinant chimeric antigen polypeptide is produced by further comprising: removing the one or more new epitopes from at least one heterologous amino acid sequence or amino acid residues derived from the second species or an additional species after inserting one or more new epitopes into a portion of the amino acid sequence of the polypeptide derived from the first species, linking to the portion, creating in the portion, or replacing or substituting the portion.

36. The method according to any one of claims 14 to 35, wherein at least two or more different heterologous amino acid sequences or amino acid residues are inserted into, linked to, created in, or replaced or superseded from the amino acid sequence of the polypeptide derived from the first species.

37. The method of claim 36, wherein the at least two or more different heterologous amino acid sequences or amino acid residues are derived from different animal species.

38. The method of claim 37, wherein at least one of the at least two or more different heterologous amino acid sequences or amino acid residues is derived from humans, and at least one of the at least two or more different heterologous amino acid sequences or amino acid residues is derived from non-human or animal species.

39. The method according to any one of claims 36 to 38, wherein at least one of the heterologous amino acid sequences or amino acid residues comprises an artificial epitope not derived from the at least one second species.

40. The method according to any one of claims 36 to 39, wherein at least one of the heterologous amino acid sequences or amino acid residues comprises an epitope originally derived from the at least one second species, which is immune-silenced in the first species (cannot generate an antibody response in the first species), but is modified to become an immune-active epitope, which can generate an antibody response against the first species.

41. The method according to any one of claims 36 to 40, wherein at least one novel epitope in the heterologous amino acid sequence or amino acid residues is modified such that the antibody generated by the first species against the modified novel epitope binds with lower strength or more slowly compared to a comparable unmodified novel epitope.

42. The method according to any one of claims 14 to 41, wherein the recombinant chimeric antigen polypeptide further comprises at least one novel epitope derived from at least a second species, the second species being dissimilar to the first species, and the at least one novel epitope being capable of generating antibodies against the first species.

43. The method according to any one of the preceding claims, or the method according to any one of claims 1 to 42, wherein a portion of the plurality of polypeptides comprises an amino acid sequence from the same species as the host, and the plurality of epitopes are from a different species than the host.

44. The method according to any one of the preceding claims, or the method according to any one of claims 1 to 43, wherein the host produces antibodies against each of the plurality of epitopes at substantially similar titers.

45. The method according to any one of the preceding claims, or the method according to any one of claims 1 to 44, wherein the portions of the plurality of polypeptides are derived from rabbits, and the plurality of epitopes are derived from species other than rabbits.

46. ​​The method according to any one of the preceding claims, or the method according to any one of claims 1 to 45, wherein the portion of the plurality of polypeptides is derived from the rabbit CDv6 polypeptide.

47. The method according to any one of the preceding claims, or the method according to any one of claims 1 to 45, wherein the portion of the plurality of polypeptides is derived from the rabbit myoglobin polypeptide.

48. A method for screening or identifying the presence of antibodies or multiple antibodies specific to a polypeptide antigen in a sample, the method comprising: (a) Provide a sample, wherein optionally the sample comprises a biological fluid, and optionally the biological fluid comprises a serum or blood sample or ascites, and optionally the serum, blood sample or ascites is harvested from a non-human animal immunized with a plurality of polypeptides, wherein each of the plurality of polypeptides contains one or more epitopes present in the antigen. (b) Provide a solid or semi-solid surface on which one, several or all of the plurality of polypeptides are attached, wherein optionally the solid or semi-solid surface is a bead, pore, biochip, microchip, column, tube or capillary, or microfluidic chip or device. (c) wherein the antibody specific to the polypeptide antigen or the plurality of antibodies are specifically bound by one, several or all of the plurality of polypeptides, the sample is brought into contact with the solid or semi-solid surface or the sample is passed through the solid or semi-solid surface. as well as (d) Identify whether one or more antibodies specific to the polypeptide antigen have specifically bound to one, several, or all of the various polypeptides attached to the solid or semi-solid surface.

49. The screening method of claim 48, the method further comprising separating the antibody or the plurality of antibodies specific to the polypeptide antigen by eluting the antibody or the plurality of antibodies that specifically bind the plurality of polypeptides from the surface of the solid or semi-solid.

50. The screening method according to claim 48 or 49, wherein the plurality of polypeptides includes the plurality of polypeptides used in the method for generating a balanced immune response according to any one of claims 1 to 47.

51. A composition comprising a plurality of polypeptides, wherein each polypeptide comprises one epitope or subset of the plurality of epitopes present in the antigen.

52. The composition of claim 51, wherein at least one of the plurality of polypeptides is engineered to remove one, several or all of the dominant epitopes from the antigen, or has been modified such that one or more dominant epitopes are no longer immunogenic in the first species, or have significantly reduced immunogenicity in the first species.

53. The composition according to claim 52, wherein a significant reduction in immunogenicity means a reduction in immunogenicity of at least about 85%, 90%, or 95%.

54. The composition according to any one of claims 51-53, wherein the antigen comprising a plurality of epitopes comprises a protein, optionally a recombinant chimeric protein.

55. The composition of claim 53, wherein the recombinant chimeric antigen polypeptide comprising multiple epitopes comprises: (a) Polypeptides derived from the first species, and (b) at least one epitope comprising a heterologous amino acid sequence or amino acid residue derived from at least a second species. At least one heterologous amino acid sequence or amino acid residue derived from the second species is inserted into, linked to, created in, or replaces or substitutes the amino acid sequence of the polypeptide derived from the first species; Furthermore, the amino acid sequence of the recombinant chimeric antigen polypeptide is essentially composed of amino acid sequences derived from the first species. Furthermore, when the amino acid sequence from the second species is inserted into, linked to, created in, or replaces or substitutes for a portion of the amino acid sequence of the polypeptide from the first species, at least one new epitope is generated, formed, or created on the polypeptide from the first species. When the recombinant chimeric antigen polypeptide is applied to the first species, the new epitope enables the first species to generate a humoral antibody response specific to the at least one new epitope. When the recombinant chimeric antigen polypeptide is used to generate a humoral immune response from an animal of the first species, the polyclonal antibody generated in the first species binds substantially only specifically to the at least one novel epitope and substantially nonspecifically to a polypeptide derived from the first species that lacks the at least one novel epitope, or an epitope created, formed, or generated by the at least one heterologous amino acid sequence or amino acid residue derived from the second or additional species, wherein the at least one heterologous amino acid sequence or amino acid residue is inserted into, linked to, created in, or replaces or substitutes the portion of the polypeptide derived from the first species.

56. The composition of claim 55, wherein the recombinant chimeric antigen polypeptide comprising multiple epitopes derived from the second species is a homolog of the polypeptide derived from the first species.

57. The composition of claim 55, wherein the amino acid sequence from the at least one second species is homologous to the first species and is inserted into a portion of the amino acid sequence of the polypeptide derived from the first species, linked to the portion, created in the portion, or replaces or substitutes the portion or the at least one homologous second species sequence, replacing all or almost all structurally homologous segments or portions of the amino acid sequence of the polypeptide derived from the first species.

58. The composition of claim 55, wherein the amino acid sequence of the at least one second species is homologous to the first species, and the at least one homologous second species sequence inserted into, linked to, created in, or replacing the portion of the amino acid sequence of the polypeptide derived from the first species is structurally homologous to the amino acid sequence of the polypeptide derived from the first species.

59. The composition of claim 55, wherein the homolog of the first species has at least about 25% to 99% sequence identity with its homolog in the second species.

60. The composition of claim 55, wherein the homolog of the first species has substantially the same secondary and / or tertiary structure as its homolog in the second species.

61. The composition according to claims 59 and 60, wherein the homolog of the first species has at least about 25% to 99% sequence identity with its homolog in the second species and has substantially the same secondary and / or tertiary structure as its homolog in the second species.

62. The composition of claim 55, wherein the homolog of the first species has at least about 50% sequence identity with its homolog in the second species.

63. The composition of claim 62, wherein the homolog of the first species has at least about 70% sequence identity with its homolog in the second species.

64. The composition of claim 62, wherein the homolog of the first species has at least about 80% sequence identity with its homolog of the second species.

65. The composition of claim 62, wherein the homolog of the first species has at least about 90% sequence identity with its homolog in the second species.

66. The composition according to any one of claims 55 to 65, wherein the amino acid sequence from the first species and the amino acid sequence from the second species have a Z score of about 2 to about 8 when aligned using a distance matrix alignment.

67. The composition of claim 66, wherein the amino acid sequence from the first species and the amino acid sequence from the second species have a Z score of at least 8 when aligned using a distance matrix alignment.

68. The composition according to any one of claims 55 to 67, wherein the polypeptide derived from the first species and its homologous polypeptide derived from the second species are antibodies.

69. The composition according to any one of claims 55 to 67, wherein the polypeptide derived from the first species and the at least one heterologous amino acid sequence derived from the second species are both derived from the antibody heavy chain or the antibody light chain.

70. The composition of claim 69, wherein the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain, or the light chain is a κ or λ light chain.

71. The composition according to any one of claims 55-70, wherein the first species is a mammal species; the second animal is a mammal species; or, the first species is an animal of the order Galliformes or family Phasianidae, and the second species is a mammal species.

72. The composition according to any one of claims 55-70, wherein the first species is a rabbit, a rodent species, a sheep, a goat, a pig, a cow, a horse, or a chicken; and the second species is a human.

73. The composition according to claim 72, wherein the rodent species is a rat or a mouse.

74. The composition according to any one of claims 55-73, wherein at least about 80% to about 99% of the amino acid sequence of the recombinant chimeric antigen polypeptide is an amino acid sequence derived from the first species, and / or about 1% to about 20% of the amino acid sequence of the recombinant chimeric antigen polypeptide is an amino acid sequence derived from the at least one second species.

75. The composition according to any one of claims 55-74, wherein one, two, three, four, five, six, seven, eight or more dominant epitopes are removed or deleted, or modified such that they are no longer immunogenic in the first species, or their immunogenicity in the first species is significantly reduced.

76. The composition according to any one of claims 55-75, wherein after inserting, linking, creating in, replacing or substituting the portion of the amino acid sequence of the polypeptide derived from the first species, the one or more new epitopes have been removed from at least one heterologous amino acid sequence or amino acid residue derived from the second species or an adjoint species.

77. The method according to any one of claims 55 to 76, wherein at least two or more different heterologous amino acid sequences or amino acid residues have been inserted into, linked to, created in, replaced or substituted for a portion of the amino acid sequence of the polypeptide derived from the first species.

78. The composition according to claim 77, wherein the at least two or more different heterologous amino acid sequences or amino acid residues are derived from different animal species.

79. The composition of claim 78, wherein at least one of the at least two or more different heterologous amino acid sequences or amino acid residues is derived from humans, and at least one of the at least two or more different heterologous amino acid sequences or amino acid residues is derived from non-human or animal species.

80. The composition according to any one of claims 55-79, wherein at least one of the heterologous amino acid sequences or amino acid residues comprises an artificial epitope not derived from the at least one second species.

81. The composition according to any one of claims 77-80, wherein at least one of the heterologous amino acid sequences or amino acid residues comprises an epitope originally derived from the at least one second species, which is immune-silenced in the first species (cannot generate an antibody response in the first species), but is modified to become an immune-active epitope, which can generate an antibody response against the first species.

82. The composition according to any one of claims 77-81, wherein at least one novel epitope of the heterologous amino acid sequence or amino acid residues is modified such that the antibody generated by the first species against the modified novel epitope binds with lower strength or more slowly compared to a comparable unmodified novel epitope.

83. The composition according to any one of claims 55-82, wherein the recombinant chimeric antigen polypeptide further comprises at least one novel epitope derived from at least a second species, the second species being dissimilar to the first species, and the at least one novel epitope being capable of generating antibodies against the first species.

84. The composition according to any one of claims 51-83, wherein a portion of the plurality of polypeptides comprises an amino acid sequence from the same species as the host, and the plurality of epitopes originate from a different species than the host.

85. The composition according to any one of claims 51-84, wherein the host produces antibodies against each of the plurality of epitopes at substantially similar titers.

86. The composition according to any one of claims 51-85, wherein the portions of the plurality of polypeptides are derived from rabbits, and the plurality of epitopes are derived from species other than rabbits.

87. The composition according to any one of claims 51-86, wherein the portion of the plurality of polypeptides is derived from the rabbit CDv6 polypeptide.

88. The composition of any one of claims 51-87, wherein the portion of said plurality of polypeptides is derived from rabbit myoglobin polypeptide.

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