Patent Application
20230105415
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 416,572 byte ASCII (text) file named “SeqList” created on Feb. 21, 2021.
The disclosure relates to immunoglobulin variants that induce potent immune responses without an adjuvant and vectors for producing such variants in plants.
Subunit vaccines consisting of recombinant protein antigens are very promising due to their safety, ease of production, and capacity to elicit targeted immune responses tailored towards desired epitopes. When delivered by themselves, however, these antigens often fail to generate robust and long-lasting immune responses, necessitating strategies to enhance their immunogenicity. Accordingly, recombinant protein antigens have been candidates for therapeutic uses. Protein fusions to the immunoglobulin Fc domain have demonstrated tremendous potential as therapeutic candidates. Fusion of a protein of interest to Fc can enhance the solubility and stability of the fusion partner while also allowing simple and cost-effective purification via protein A/G affinity chromatography. Furthermore, by interacting with neonatal Fc receptors (FcRn) in the body, Fc-fusions can escape lysosomal degradation, thereby extending the serum half-life of the Fc-fusion.
While much of the work with Fc-fusions has focused on improving their therapeutic potential, fewer studies have investigated Fc-fusion as a strategy to enhance antigen immunogenicity. Antigen-presenting cells containing Fcγ receptors and the complement receptor C1q can uptake and process IgG-bound antigen. However, these interactions require high avidity binding for activation, and thus monovalent Fc-antigen fusions cannot efficiently utilize these pathways. On the other hand, larger antigen-antibody immune complexes with multivalent Fc domains can cross-link Fc receptors and efficiently bind C1q, resulting in greatly improved uptake and presentation by dendritic cells, as well as improved activation of T-cells. Immune complexes generated by mixing antibody with antigen often yields inconsistent results: immune responses may be skewed towards favorable antigenic sites, but overall immunogenicity may not be markedly improved.
By contrast, recombinant immune complexes (RICs), have been used to produce vaccine candidates for Clostridium tetani, Ebola virus, Mycobacterium tuberculosis, dengue virus, and human papillomavirus. An RIC is an IgG genetically fused to its cognate antigen, which allow the formation of larger highly immunogenic antigen-antibody clusters that mimic those found during native infection.
This disclosure is directed to recombinant proteins that are immunoglobulin variants, for example immunoglobulin G (IgG) variants, and their methods of production and use. In some aspects, the IgG variants are based on the 6D8 antibody. In certain embodiments where the parts of antibody in the recombinant protein is the 6D8 antibody, the Fc fusion, which comprises the CH2 domain and the CH3 domain of the antibody, comprises the substitution mutations E345R, E430G, and S440Y.
In some embodiments, the recombinant protein comprises a unit of an antigen, a unit of a Fc fusion comprising a CH2 domain and a CH3 domain of an IgG, a unit of a variable heavy chain (VH) domain of the IgG, a unit of a CH1 domain of the IgG; and a unit of a light chain variable (VL) domain of the IgG. The unit of the antigen is not an epitope of the IgG, and it is linked to the unit of the VH domain of the IgG at the N-terminus or to the CH3 domain of IgG at the C-terminus. The unit of the CH1 domain of IgG is linked to the CH2 domain of the IgG, while the unit of the VL domain of the IgG is fused to the unit of the VH domain of the IgG and to the CH1 domain of the IgG. In some implementations, the recombinant protein further comprises a unit of an epitope tag, wherein the epitope tag is an epitope of the IgG.
In some aspects, the recombinant protein comprises two units of the antigen, two units of the Fc fusion, two units of the CH1 domain of the IgG, two units of the VH domain of the IgG, and two units of the VL domain of the IgG. A disulfide bond formed at the linkage of the CH2 domain and the CH1 domain of the IgG links the two units of the Fc fusion. In some aspects, the each unit of the VL domain of the IgG is linked to a unit of VH domain of the IgG and the CH1 domain of the IgG. In particular implementations, the recombinant protein does not comprise any light chain constant (CL) domain of the IgG. In some embodiments of the recombinant protein comprising two units of the antigen, two units of the Fc fusion, two units of the CH1 domain of the IgG, two units of the VH domain of the IgG, and two units of light chain variable (VL) domain of the IgG, the recombinant protein further comprises two units of an epitope tag, wherein the epitope tag is an epitope of the IgG. In some implementations, the two units of the epitope tag are linked to the two units of the Fc fusion at the C-terminus of the CH3 domain of the IgG, while the two units of the antigen are linked to the two units of the VH domain of the IgG. In other implementations, the two units of the epitope tag are linked to the two units of the antigen, while the two units of the antigen are linked to the two units of Fc fusion at the C-terminus of the CH3 domain of the IgG. In particular embodiments, the antibody is the 6D8 antibody and the epitope tag comprises the peptide sequence YKLDIS (SEQ ID NO. 1).
In other embodiments, the recombinant protein comprises a unit of an antigen, a unit of a Fc fusion comprising a CH2 domain and a CH3 domain of an IgG, a unit of a VH domain of the IgG, a unit of a CH1 domain of the IgG, a unit of a VL domain of the IgG; and a unit of a CL domain of the IgG. In such embodiments, the IgG is the 6D8 antibody, and the Fc fusion comprises the substitution mutations E345R, E430G, and S440Y. The unit of the antigen is not an epitope of the 6D8 antibody, and it is linked to the CH3 domain of IgG at the C-terminus. The unit of the VL domain of the IgG is linked to the unit of the CL domain of the IgG, and the unit of the CL domain of the IgG is linked to the CH1 domain of the IgG. The unit of the CH1 domain of IgG is then linked to the CH2 domain of the IgG. In some aspects, the recombinant protein further comprises an epitope tag for the 6D8 antibody, for example comprising the peptide sequence YKLDIS (SEQ ID NO. 1).
In some aspects, the epitope tag in the recombinant protein comprises the sequence VYKLDISEA (SEQ ID NO. 2). In other aspects, epitope tag consists of the sequence YKLDIS (SEQ ID NO. 1).
In still other embodiments of the recombinant protein, IgG variant comprises two units of an antigen, two units of the Fc fusion, two units of a VH domain of the IgG, and two unit of a CH1 domain of the IgG. The antigen is not an epitope of the IgG, and the two units of the antigen are linked to the two units of the VH domain of the IgG at the N-terminus. The two units of the CH1 domain of IgG are linked to the two units of the Fc fusion at the CH2 domain of the IgG. In some aspects, the recombinant protein does not comprise a CL domain of the IgG and does not comprise a VL domain of the IgG.
In certain implementations, the antigen of the recombinant protein is from Zika virus, for example, the unit of the antigen comprises K301-T406 of Accession No. AMC13911.1. In other implementations, the antigen of the recombinant protein is from norovirus, for example, the unit of the antigen comprises at least one portion from the major capsid protein of noroviruses and/or at least one portion from nonstructural protein 1 (NS1) of noroviruses. In some embodiments, the unit of the antigen comprises at least one 5- to 500-residue long portion from the protruding domain of the norovirus major capsid protein, the shell domain of the norovirus major capsid protein, or from NS1 of noroviruses. In some aspects, the antigen of the recombinant protein may comprise a plurality of antigenic peptides. For example, in some implementations, the unit of the antigen comprises at least one portion from the major capsid protein or at least one portion from NS1 of a plurality of norovirus strains or species, for example, from human norovirus virus GI.3, human norovirus virus GII.4, and murine norovirus (MNV). In some embodiments, the unit of the antigen comprises at least one sequence set forth in SEQ ID NOs. 34-72.
The methods of production described herein comprise expressing the recombinant protein in a plant. In other words, the recombinant protein is produced in a transgenic plant. In certain implementations, the recombinant protein is expressed in a transgenic plant silenced for xylosyltransferase and fucosyltransferase. In some aspects, the method of producing the recombinant protein described herein comprises introducing into agrobacteria a vector selected from the group consisting of: pBYR11eM-h6D8ZE3, pBYR11eMa-BAZE3-Hgp371, pBYR11eMa-BAZE3-H, pBYKEMd-HZE3, pBYKEMd-ZE3H, pBYKEMd-ZE3Hx, pBYKEMd-HVLZe, pBYKEMd2-HVL-Hx, pBYKEMd2-HVLZnt, pBYKEMd2-ZHVLnt, pBYKEMd2-ZHVLe, pBYKEMd2-ZHVLhx, pBYKEAM-N12MHd, pBYKEAM-NPHd, pBYKEAM-NSHd, pBYKEAM-NTHd, pBYKEHM-CPHd, pBYKEHM-CSHd, and pBYKEHM-CTHd and infiltrating a plant part with agrobacteria containing the vector to produce a transformed plant part. Crude protein is then extracted from the transformed plant followed by purification for the recombinant protein. For methods of production where the vector introduced into agrobacteria is selected from the group consisting of: pBYR11eM-h6D8ZE3, pBYR11eMa-BAZE3-Hgp371, pBYR11eMa-BAZE3-H, and pBYKEMd-HZE3, the method further comprises co-infiltration with agrobacteria containing pBYKEMd-6D8K and agrobacteria containing the vector.
The methods of use described herein include a method of inducing an immune response in a subject against the antigen in the recombinant protein. The method comprises administering the described recombinant protein to the subject. In certain embodiments, the method induces in the subject an immune response against Zika virus. In such embodiments, the recombinant protein comprises an antigen with an amino acid sequence comprising K301-T406 of Accession No. AMC13911.1. In other embodiments, the method induces in the subject an immune response against norovirus, including human norovirus and MNV. In such embodiments, the recombinant protein comprises an antigen from the VP1 of a norovirus, for example from its protruding domain or the shell domain, or from NS1 of a norovirus.
In some implementation, the subject is administering a composition comprising a recombinant protein with an antigen from VP1 of a plurality of noroviruses and a recombinant protein with an antigen from NS1 of a plurality of noroviruses. In still other implementations, the recombinant protein is produced in a transgenic plant. In some aspects, the transgenic plant is silenced for xylosyltransferase and fucosyltransferase.
Detailed aspects and applications of the disclosure are described below in the following drawings and detailed description of the technology. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.
In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that embodiments of the technology disclosed herein may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices and technologies to which the disclosed technologies may be applied. The full scope of the technology disclosed herein is not limited to the examples that are described below.
The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.
As used herein, the term “6D8 antibody” refers to a monoclonal antibody against the GP1 protein of Ebola virus (described in Wilson et al., 2000; plant optimized sequence described in Huang et al., 2010). In some embodiments, the CH1 domain of the 6D8 antibody refers to a peptide sequence comprising TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO. 5) or a peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence similarity. In some embodiments, the CH2 domain of the 6D8 antibody refers to a peptide sequence comprising DKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALAPIEKTISKAKG (SEQ ID NO. 6) or a peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence similarity. In some embodiments, the CH3 domain of the 6D8 antibody refers to a peptide sequence comprising QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO. 7). In some embodiments, the variable heavy chain domain of the 6D8 antibody refers to a peptide sequence comprising GenBank Accession No. AEB96146 or a peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence similarity. In some embodiments, the variable light chain domain of the 6D8 antibody refers to a peptide sequence comprising GenBank Accession No. AEB96146 or a peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence similarity. In some aspects, nucleic acid encoding the light chain variant domain is set forth in GenBank Accession No. HQ407546. In some aspects, the nucleic acid encoding the heavy chain variant domain is set forth in GenBank Accession No. AEB96148.
As used herein, the term “linked” when used to describe a protein structure or configuration of protein domains refers to a linkage between two domains or structural portions, preferably where there are no other intervening domains or structural portions except a linker. In some aspects, the term “linker” as used herein refers to an amino acid sequence between protein domains or structural portions where its only function is to link the protein domains or structural portions. For example, the linker is sequence consisting of glycine, valine, and/or threonine residues. In some embodiments, the linker is sequence consisting of glycine, valine, threonine, alanine, and/or serine residues. In some aspects, the linker consists of 1 to 50 amino acids in length, for example, 3, 4, 6, 10, 12, 14, or 16 amino acids in length. In certain embodiments, the linker is a flexible linker.
As used herein, the term “unit” refers to a single peptide that is a functional fragment of a larger multi-component protein. The single peptide may be an antigenic fragment or a domain of a protein, for example a domain of an immunoglobulin. Accordingly, as used herein, the description of a recombinant protein comprising two units of a peptide refers to the recombinant protein having two such functional fragments that may or may not be linked together, for example, a dimer of the peptide.
As used herein, the term “immune complex” refers to a complex comprising immunoglobulin molecules or fragments thereof bound to its cognate antigen. As used herein, the term “recombinant immune complex” or “RIC” refers to an immune complex that is not produced by the original species that naturally produces the immunoglobulin molecule in the immune complex. For example, an exemplary recombinant immune complex comprises human immunoglobulin, which is synthesized in plants.
The disclosure relates to immunoglobulin variants. In particular preferred embodiments, the disclosure is directed to recombinant proteins that are variants of immunoglobulin G, also referred to herein as IgG fusions. In spite of the deviation from the natural structure of immunoglobulins, the described recombinant proteins do have immunogenic potential. In fact, the immune response induced by the recombinant protein is the same kind of immune response expected of the antigen in the recombinant protein. The recombinant proteins described herein are suitable for creating vaccines for targeting a variety of pathogens, for example Zika virus and norovirus.
Regardless of the variations in immunoglobulin structure, glycosylation state of the Fc strongly controls its function. By modulating the stability, conformation, and aggregation of the Fc, glycosylation can enhance or inhibit binding to Fcγ receptors, FcRn, and C1q. These alterations result in important differences in antibody effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody-dependent enhancement of viral infection (ADE). Advances in glycoengineering have allowed targeted optimization of the Fc glycosylation state in a variety of recombinant expression systems. Using glycoengineered plants lacking core xylose and fucose N-glycans, an anti-CD20 antibody was produced with improved binding to FcγRI, FcγRIIIa, and C1q. The antibody had enhanced ADCC and CDC compared to a commercial anti-CD20 antibody produced in mammalian cells. Similarly, anti-DENV antibodies produced in glycoengineered plants have been shown to forgo their ADE activity and, consequently, have superior efficacy and safety profiles than their mammalian cell-produced counterparts. Antibody therapeutics made in glycoengineered plants have been used to treat rhesus macaques and humans with Ebola, HIV, and Chikungunya virus disease.
Mutations in the Fc region have been identified that confer desirable properties to antibodies. Introduction of M252Y, S254T, and T256E mutations increased the serum half-life of an anti-respiratory syncytial virus antibody from 20 days to 60 days in humans by improving interactions with FcRn under low pH conditions. An H237Y mutation reduced detrimental cleavage of the hinge region while improving FcγRIII binding and ADCC activity. Engineering additional disulfide bonds has been reported to prevent unfolding and aggregation of Fc-fusions. S239D and I332E mutations improved FcγRIII binding and ADCC activity of a CD37 antibody. Replacing the IgG1 hinge region with the longer hinge region from IgG3 or from a camelid antibody increased ADCC of an epidermal growth factor receptor antibody. An IgG1 with E345R, E430G, and S440Y mutations formed hexamers that had greatly enhanced C1q binding and complement activation. The mutations T437R and K248E also promoted multimerization and improved effector functions of an OX40 receptor antibody.
The IgG variants described herein can be properly assembled with human-like glycosylation and expressed at very high levels in plants. These constructs can be efficiently made in plants, assemble appropriately, and purified via protein G column chromatography. As shown in the examples, the purified recombinant proteins are potently immunogenic.
As shown in the examples described herein, optimizing various aspects of the antibody design, such as complex size, to produce the described immunoglobulin variants resulted in optimized expression levels in plant and improved stability, solubility, and immune receptor binding. In certain embodiments where the immunoglobulin variants are designed with a portion of the Zika virus envelope protein domain III as the antigen, total IgG titers raised by the administration of the immunoglobulin variant was 150-fold higher compared to the total IgG titer raised by administration of the antigen alone. In fact, the endpoint titers was >1:500,000 with administration of only two doses of the immunoglobulin variant without any adjuvant. The recombinant proteins could be produced at levels exceeding the estimated thresholds for commercial viability of antibody therapeutics.
The recombinant proteins described herein are variant immunoglobulin structures comprising a unit of an antigen, a unit of a Fc fusion, a unit of a variable heavy chain (VH) domain of the IgG, and a unit of a CH1 domain of the IgG. The Fc fusion comprises a CH2 domain and a CH3 domain of an IgG. Protein fusions to the immunoglobulin Fc domain are highly successful therapeutics. They can enhance the solubility and stability of the fusion partner, while also providing a means for simple and cost-effective purification. Furthermore, by interacting with neonatal Fc receptors (FcRn) in the body, Fc-fusions can escape lysosomal degradation, thereby extending the serum half-life of the Fc-fusion. The recombinant protein induces a greater immune response against the antigen attached to the recombinant protein than the antigen alone. Thus, the recombinant protein increases the immunogenicity of the antigen.
The unit of the antigen is not an epitope of the IgG. In certain embodiments, the unit of the antigen is from a different organism than the epitope of the IgG. The amino acid sequence of the unit of the antigen is between 5 and 500 residues long. In certain embodiments, the unit of the antigen is between 5 and 400 residues long. For example, the unit of the antigen is between 5 and 300 residues, between 5 and 250 residues, between 5 and 200 residues, between 5 and 100 residues, between 9 and 300 residues, between 9 and 250 residues, between 9 and 200 residues, between 9 and 100 residues, between 10 and 300 residues, between 10 and 250 residues, between 10 and 200 residues, between 10 and 100 residues, between 20 and 300 residues, between 20 and 250 residues, between 20 and 200 residues, between 20 and 100 residues, between 30 and 300 residues, between 30 and 250 residues, between 30 and 200 residues, between 30 and 100 residues, between 50 and 300 residues, between 50 and 250 residues, between 50 and 200 residues, or between 50 and 100 residues in length. In some aspects, the unit of the antigen comprises tandem-linked epitopes.
In some aspects, the tandem-linked epitopes comprise different epitopes, which may or may not be from the same or similar antigen protein. For example, a recombinant protein that targets a plurality of norovirus can comprise epitopes on the same target antigen from the plurality of noroviruses (see recombinant immune complexes studied in
The unit of the antigen is linked to the unit of the VH domain of the IgG at the N-terminus or to the CH3 domain of IgG at the C-terminus. The unit of the CH1 domain of IgG is linked to the CH2 domain of the IgG. In some aspects, the unit of an antigen, the unit of the Fc fusion, the unit of the VH domain of the IgG, and the unit of a CH1 domain of the IgG forms half of the recombinant protein. For example, the recombinant protein self assembles upon production wherein a disulfide bond is formed at the linkage of the CH2 domain and the CH1 domain to link two units of the Fc fusion (see
In certain embodiments, the recombinant protein comprises a unit of an antigen, a unit of a Fc fusion comprising a CH2 domain and a CH3 domain of an IgG, a unit of a VH domain of the IgG, a unit of a CH1 domain of the IgG; and a unit of a light chain variable (VL) domain of the IgG. The unit of the antigen is not an epitope of the IgG, and it is linked to the unit of the VH domain of the IgG at the N-terminus or to the CH3 domain of IgG at the C-terminus. The unit of the CH1 domain of IgG is linked to the CH2 domain of the IgG, while the unit of the VL domain of the IgG is fused to the unit of the VH domain of the IgG and to the CH1 domain of the IgG.
In some aspects, the recombinant protein is self-assembled upon production, and a disulfide bond formed at the linkage of the CH2 domain and the CH1 domain of the IgG links the two units of the Fc fusion. Accordingly, the recombinant protein comprises two units of the antigen, two units of the Fc fusion, two units of the CH1 domain of the IgG, two units of the VH domain of the IgG, and two units of the VL domain of the IgG. In some aspects, the each unit of the VL domain of the IgG is linked to a unit of VH domain of the IgG and the CH1 domain of the IgG. In particular implementations, the recombinant protein does not comprise any light chain constant (CL) domain of the IgG. The recombinant protein of such embodiments is a single-chain antibody variant (see for example,
In some embodiments of the recombinant protein comprising two units of the antigen, two units of the Fc fusion, two units of the CH1 domain of the IgG, two units of the VH domain of the IgG, and two units of the VL domain of the IgG, the recombinant protein further comprises two units of an epitope tag, wherein the epitope tag is an epitope of the IgG. In some implementations, the two units of the epitope tag are linked to the two units of the Fc fusion at the C-terminus of the CH3 domain of the IgG, and the two units of the antigen are linked to the two units of the VH domain of the IgG. In other implementations, the two units of the epitope tag are linked to the two units of the antigen, and the two units of the antigen are linked to the two units of Fc fusion at the C-terminus of the CH3 domain of the IgG. Where the antibody is the 6D8 antibody, the epitope tag comprises the peptide sequence YKLDIS (SEQ ID NO. 1). In some aspects, the epitope tag comprises the sequence VYKLDISEA (SEQ ID NO. 2). In other aspects, the epitope tag consists of the sequence YKLDIS (SEQ ID NO. 1).
In other embodiments, the recombinant protein comprises a unit of an antigen, a unit of a Fc fusion comprising a CH2 domain and a CH3 domain of an IgG, a unit of a VH domain of the IgG, a unit of a CH1 domain of the IgG, a unit of a VL domain of the IgG; and a unit of a CL domain of the IgG. In such embodiments, the IgG is the 6D8 antibody, and the Fc fusion comprises at least one substitution mutation selected from the group consisting of: E345R, E430G, and S440Y. In certain embodiments, the Fc fusion comprises the substitution mutations E345R, E430G, and S440Y. The unit of the antigen is not an epitope of the 6D8 antibody, and it is linked to the CH3 domain of IgG at the C-terminus. The unit of the VL domain of the IgG is linked to the unit of the CL domain of the IgG, and the unit of the CL domain of the IgG is linked to the CH1 domain of the IgG. The unit of the CH1 domain of IgG is then linked to the CH2 domain of the IgG. In some aspects, the recombinant protein further comprises an epitope tag for the 6D8 antibody, for example comprising the peptide sequence YKLDIS (SEQ ID NO. 1). In some aspects, the epitope tag in the recombinant protein comprises the sequence VYKLDISEA (SEQ ID NO. 2). In other aspects, epitope tag consists of the sequence YKLDIS (SEQ ID NO. 1).
In still other embodiments of the recombinant protein, IgG variant comprises two units of an antigen, two units of the Fc fusion, two units of a VH domain of the IgG, and two unit of a CH1 domain of the IgG. The antigen is not an epitope of the IgG, and the two unit of the antigen are linked to the two units of the VH domain of the IgG at the N-terminus. The two units of the CH1 domain of IgG are linked to the two units of the Fc fusion at the CH2 domain of the IgG. In some aspects, the recombinant protein does not comprise a CL domain of the IgG and does not comprise a VL domain of the IgG.
Zika virus (ZIKV) is a substantial global health threat that lacks safe, affordable, and efficacious vaccines. In addition to their widespread therapeutic value, IgG fusions are promising vaccine candidates due to their safety and self-adjuvating nature.
In exemplary embodiments, some IgG variants were designed to generate an immune response against ZIKV. Accordingly, the antigen in the recombinant protein is an antigen targeting ZIKV. In some aspects, the antigen targets Zika virus envelope domain III (ZE3). These IgG variants were highly stable and highly expressed in plants. Accordingly, the recombinant proteins described herein are vaccine candidates, for example against ZIKV.
In some aspects, the antigen targeting ZE3 comprises K301-T406 of Accession No. AMC13911.1. In other aspects, the antigen targeting ZE3 comprises a peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence similarity to K301-T406 of Accession No. AMC13911.1. In still other aspects, the antigen targeting ZE3 comprises a sequence that is a functionally equivalent version of corresponding regions of GenBank Accession No. AMC13911 from other strains of Zika virus, for example, the corresponding sequences of ZE3 in GenBank Accession Nos. AY632535, KU321639, KJ776791, KF383115, KF383116, KF383117, KF383118, KF383119, KF268948, KF268949, KF268950, EU545988, KF993678, JN860885, HQ234499, KU501215, KU501216, KU501217.
The recombinant proteins described herein targeting ZIKV elicited strong immune responses against ZIKV without adjuvants and only two doses (
Constructs expected to form polymeric structures were the most resistant to degradation and had the highest overall yield. By optimizing intermolecular binding of RIC, smaller complexes were generated that had greatly improved solubility, yielding 1.5 milligrams IgG fusion per gram leaf fresh weight (mg/g LFW), and equivalent or improved immunogenic properties. When used to immunize mice, the IgG fusions elicited high titers of Zika-specific antibodies using only two doses without adjuvant, exceeding the titers produced by ZE3 alone by 20-fold to 150-fold, and potently neutralized Zika virus. The IgG fusions were also found to strongly enhance IgG2a production compared to unfused ZE3 antigen in a manner that correlated with C1q binding. These findings demonstrate the excellent potential of IgG fusions as self-adjuvanting subunit vaccines for Zika virus that can be made efficiently in plants.
C1q binding of the IgG fusions targeting ZIKV was enhanced by ZE3 fusion to the IgG N-terminus. The removal of the IgG light chain (HVL and HVL-related constructs) or Fab regions also enhanced C1q binding. The addition of hexamer-inducing mutations in the IgG Fc region enhanced C1q binding as well. C1q binding was also enhanced by adding a self-binding epitope tag to create RIC or producing IgG fusions in plants that lack plant-specific (31,2-linked xylose and α1,3-linked fucose N-glycans (for example, by silencing xylosyltransferase and fucosyltransferase, see
In some aspects, the RIC construct is not preferred, because the large complex size renders them poorly soluble upon extraction (
The HVLZe fusion had slightly higher C1q binding than traditional RIC (
Table 1 summarizes characteristics of the recombinant protein, an antigen targeting ZIKV. For expression, only the yield (mg/g LFW) of the fully assembled product is shown. A greater number of “+” symbols indicates either a statistically significant increase in the mean value for that property (for C1q, IgG, and IgG2a), or a repeatably observed difference (for density). For ZHx (*), peaks of both low-density and of high-density material were observed.
The widespread occurrence of human norovirus (HNoV) infections causes tremendous economic damage and disease burden, however no approved vaccines or specific treatments are available. Efforts to control HNoV are hampered by the large genetic diversity of these viruses, which continually evolve to evade natural immunity. A wealth of published antibody mapping and T-cell data demonstrates the existence of broadly conserved HNoV B-cell and T-cell epitopes which, crucially, are targetable by the human immune system. Accordingly, it would be possible to design recombinant proteins for the purpose of eliciting immune responses against broadly conserved HNoV antigenic targets.
In exemplary embodiments, IgG variants were designed to generate an immune response against norovirus (NoV). In some aspects, the antigen of the recombinant protein comprises epitopes from a plurality of noroviruses, for example from human norovirus GI.3, human norovirus GII.4, and/or murine norovirus (MNV). The antigen in the recombinant protein targeting noroviruses comprises at least one epitope from the major capsid protein of NoV (VP1) or the nonstructural protein of NoV. In some aspects, the antigen targets the protruding domain or the shell domain of VP1. Such recombinant proteins are vaccine candidates against NoV.
In certain implementations, the recombinant proteins targeting NoV may be used in combination as a vaccination composition. As shown in the examples and
In some aspects, the antigen targeting VP1 comprises 5- to 500-residue-long portion of VP1 and/or NS1. In some embodiments, the unit of the antigen comprises at least one sequence, at least three sequences, at least six sequences, at least nine sequence, at least twelve sequences, or at least fifteen sequences set forth in SEQ ID NOs. 34-72. For example, the unit of the antigen in recombinant proteins targeting VP1 comprises at least one sequence, at least two sequences, at least three sequences, at least four sequences, at least five sequences, at least six sequences, at least seven sequences, at least eight sequences, or at least nine sequences set forth in SEQ ID NOs. 34-54. In a particular embodiment, the unit of the antigen targeting the protruding domain of VP1 comprises at least one sequence, at least two sequences, at least three sequences, at least four sequences, at least five sequences, at least six sequences, at least seven sequences, at least eight sequences, or at least nine sequences selected from SEQ ID NOs. 34-44. In certain embodiments, the unit of the antigen of a recombinant protein targeting norovirus comprises the sequences set forth in SEQ ID NOs. 34-42. In another particular embodiment, the unit of the antigen of a recombinant protein targeting the shell domain of VP1 comprises at least one sequence, at least two sequences, at least three sequences, at least four sequences, at least five sequences, at least six sequences, at least seven sequences, at least eight sequences, or at least nine sequences selected from SEQ ID NOs. 45-54. In some aspects, the unit of the antigen of a recombinant protein targeting norovirus comprises the sequences set forth in SEQ ID NOs. 45-53. In still another particular embodiment, the unit of the antigen of a recombinant protein targeting NS1 comprises at least one sequence or at least two sequences selected from SEQ ID NOs. 55-57. In some aspects, the unit of the antigen of a recombinant protein targeting norovirus comprises the sequences set forth in SEQ ID NOs. 55-57. In yet another particular embodiment, the unit of the antigen of a recombinant protein designed to generate a T-cell mediated immune response comprises at least one sequence, at least two sequences, at least three sequences, at least four sequences, at least five sequences, at least six sequences, at least seven sequences, at least eight sequences, or at least nine sequences selected from SEQ ID NOs. 58-72, for example, the peptide sequence of the unit of the antigen comprises the sequences of SEQ ID NOs. 58-72.
Plant recombinant expression systems have inherent safety, high scalability, and low production costs compared to mammalian cell systems, making them particularly well suited to make IgG fusions vaccines. While previous work has shown that some IgG fusion vaccines can enhance antigen immunogenicity, the many fusion strategies that have been developed have not been directly compared, making it difficult to determine the key properties involved in creating an optimal vaccine candidate.
The methods of production described herein comprise expressing the recombinant protein in a transgenic plant. In certain implementations, the recombinant protein is expressed in a transgenic plant silenced for xylosyltransferase and fucosyltransferase. In some aspects, for example the recombinant protein is a RIC, it is preferable to produce the proteins using plants with xylosyltransferase and fucosyltransferase silenced. The method comprises introducing into agrobacteria a binary vector that expresses a unit of the antigen, a unit of the Fc fusion, a unit of the VH domain of the IgG; and a unit of the CH1 domain of the IgG. Accordingly, this binary vector encodes the heavy chain domains of the IgG. Next, a plant part is infiltrated with agrobacteria containing the binary vector to produce a transformed plant part. Crude protein from the plant part is extracted and then purified for the recombinant protein. In some aspects, the recombinant protein is purified using methods well-established in the art, for example, protein G affinity chromatography or metal affinity chromatography.
Where the recombinant protein comprises a unit of the VL domain of the IgG and a unit of the CL domain of the IgG, the method further comprises introducing into agrobacteria a different binary vector that encodes light chain domains of the IgG, namely the unit of the VL domain of the IgG and the unit of the CL domain of the IgG. The transformation of the plant part comprises co-infiltrating the plant part with agrobacteria containing the binary vector encoding the heavy chain domains of the IgG and agrobacteria containing the binary vector encoding the light chain domains of the IgG.
In particle implementations, the method of producing the recombinant protein described herein comprises introducing into agrobacteria a vector selected from the group consisting of: pBYR11eM-h6D8ZE3, pBYR11eMa-BAZE3-Hgp371, pBYR11eMa-BAZE3-H, pBYKEMd-HZE3, pBYKEMd-ZE3H, pBYKEMd-ZE3Hx, pBYKEMd-HVLZe, pBYKEMd2-HVL-Hx, pBYKEMd2-HVLZnt, pBYKEMd2-ZHVLnt, pBYKEMd2-ZHVLe, pBYKEMd2-ZHVLhx, pBYKEAM-N12MHd, pBYKEAM-NPHd, pBYKEAM-NSHd, pBYKEAM-NTHd, pBYKEHM-CPHd, pBYKEHM-CSHd, and pBYKEHM-CTHd and infiltrating a plant part with agrobacteria containing the vector to produce a transformed plant part. Crude protein is then extracted from the transformed plant followed by purification for the recombinant protein. For methods of production where the vector introduced into agrobacteria is selected from the group consisting of: pBYR11eM-h6D8ZE3, pBYR11eMa-BAZE3-Hgp371, pBYR11eMa-BAZE3-H, and pBYKEMd-HZE3, the method further comprises co-infiltrating with agrobacteria containing pBYKEMd-6D8K and agrobacteria containing the vector.
Illustrative, Non-Limiting Example in Accordance with Certain Embodiments
The disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.
A. Design and C1q Binding of IgG-Fusions with ZIKV Envelope Domain III
ZE3 is a promising subunit vaccine candidate, however it is not strongly immunogenic on its own, necessitating high antigen doses with adjuvant and repeated immunizations. A panel of human IgG1 variants was designed based on the humanized Ebola monoclonal antibody 6D8 fused to ZE3 (
It has been reported that the antibody Fab arms may play a regulatory role in complement activation by inhibiting C1q binding unless cognate antigens are bound by the antibody. In agreement with these data, the addition of soluble antigen carrying the 6D8 epitope tag improved C1q binding (
It is interesting to note that the theoretically monomeric construct HLZ (consisting of ZE3 fused to the C-terminus of 6D8 without any epitope tag) produced a small but repeatable improvement in C1q binding (
Antigen binding may induce conformational changes in the IgG, which may improve C1q binding. Mixing 6D8 with an antigen containing the 6D8 epitope produced only a small increase in C1q binding (
N-terminal ZE3 fusion to the 6D8 CH1 domain (construct ZFc) greatly improved C1q binding (
RICs are known to be immunogenic; however, they require the co-expression of a heavy and light chain in order to form the fully assembled product. This process can be simplified by creating a single chain antibody fusion. The benefit of single-chain antibody fusions is that the entire antibody-antigen fusion can be produced in a single coding sequence, thereby eliminating the need for co-expression of heavy and light chains. In addition, the new construct will still retain the variable regions of both the heavy and light chain. The core single-chain antibody contains the variable heavy regions linked to a variable light chain region that is directly fused to an antibody heavy chain (
RIC suffer from low yield of soluble product. Addition of the 6D8 epitope tag to the C-terminus of 6D8 renders the antibody mostly insoluble; however this is prevented by removal of the light chain, suggesting the insolubility arises from large complexes of antibody bound to the epitope tag (
To measure the yield of fully assembled IgG fusions, an ELISA assay was employed that first captured ZE3 and then detected human IgG. To detect cleavage of ZE3, an ELISA measuring only total IgG was also used as a comparison. When probed for both ZE3 and IgG, the highly soluble monomeric construct ZH yielded 0.83 mg fully formed product per gram leaf fresh weight (mg/g LFW), and the similar ZFc yielded 0.58 mg/g LFW (
IgG fusions were purified to >95% homogeneity using a simple one-step purification via protein G affinity chromatography. In agreement with the expression data, the more highly oligomeric constructs showed less degradation than the other constructs, and the ZFc fusion had particularly high levels of degradation (
The stability of each construct was analyzed by comparing fully formed products and degradation products on SDS-PAGE after treatment with various temperature conditions. After five freeze-thaw cycles or two weeks at 4° C., small amounts of degradation were observed with all constructs (
To investigate the immunogenicity of the IgG fusions, BALB/c mice (n=6) were immunized subcutaneously without adjuvant with two doses of each IgG variant such that the total dose of ZE3 delivered was 8 μg. As a control, mice were also immunized with 8 μg unfused plant-expressed ZE3. All IgG fusions very strongly enhanced the production of ZE3-specific antibodies, producing 20-fold to 150-fold higher total IgG titers than ZE3 alone (
F. Design, Expression, and Purification of IgG-Fusions with Norovirus Epitopes
The norovirus (NoV) capsid protein VP1 consists of an inner shell domain (S), which forms the core surrounding the viral genome, and a protruding domain (P), which is further subdivided into P1 and P2 subdomains. Traditional human norovirus (HNoV) vaccine approaches, as well as natural infection, elicit immune responses primarily targeting immunodominant epitopes on the viral capsid that are poorly conserved, resulting in limited protection against novel strains and against related viruses from different genotypes. Nearly all HNoV vaccine candidates have focused on virus-like particles (VLPs) made from the VP1 capsid protein. Based on sequence divergence of the major capsid protein VP1, ten genogroups (GI-GX) which contains over forty genotypes of NoVs have been identified. Of these ten genogroups, GI and GII cause the most human disease. Yet, even the best attempts at generating conserved VLPs have produced only modest reductions of disease severity in human clinical trials and generally struggled to induce broadly protective immunity.
One broadly reactive antibody mapped to a strongly conserved epitope at the base of P1: LPQEWVQYFYQEAAPA (SEQ ID NO. 34). Critically, this antibody binds linear epitopes in P1, and thus the native VP1 conformation is not necessary to elicit functional antibodies. Antibodies targeting this epitope bind with high affinity to VLPs from 8 GI, 13 GII, and 1 GIV genotype. Antibodies directed against a second broadly reactive, linear epitope (ALLRFVNPDTGRVLFE, SEQ ID NO. 37) bind VLPs from at least 3 GI and 7 GII genogroups. A third broadly conserved linear epitope at the base of the P1 domain is DSWVNQFYTLAP (SEQ ID NO. 41). The S domain is the most highly conserved region of VP1. Antibodies targeting the strongly conserved linear epitopes QNVIDPWIRNNF (SEQ ID NO. 45), QAPGGEFTVSPRNAPGE (SEQ ID NO. 46), and KVI FAAVPP (SEQ ID NO. 47) have been identified with broad cross-reactivity to NoV genogroups. Conserved HNoV T-cell epitopes were identified in humansl3, namely the epitope TMFPHI IVDV (SEQ ID NO. 54) in the VP1 S domain elicited broad T-cell activation against both GI and GII HNoV. In addition, a dominant T-cell epitope (SWVSRFYQL, SEQ ID NO. 64) in the P1 C-terminal domain that is highly conserved between all NoV genogroups, including HNoV, was critical for viral clearance in NoV-infected mice. This region plays a vital role in capsid assembly, and thus structural constraints limit the potential for mutational escape. The nonstructural protein NS1, which is proteolytically cleaved from NS1-2, is secreted from cells infected with murine NoV (GV) or transfected with HuNoV NS1-2. Secreted NS1 suppresses IFN-λ, production in the intestinal tract of mice. Importantly, vaccination with NS1 alone protected mice better than vaccination with P-domain, highlighting NS1 as a vaccine target. Vaccination with NS1-2 yielded antibodies directed against NS1.
For the development of antigens to make immunoglobulin variants targeting norovirus, epitopes from the protruding domain of VP1 of human norovirus GI.3, human norovirus GII.4, and murine norovirus (MNV) have been identified (SEQ ID NOs. 34-44). Also identified are epitopes from the shell domain of VP1 of GI.3, GII.4, and MNV (SEQ ID NOs. 45-54). The NS1 epitopes of MNV is set forth in SEQ ID NO. 55. The NS1 epitope of GI.3 is set forth in SEQ ID NO. 56. The NS1 epitope of GII.4 is set forth in SEQ ID NO. 57. The T-cell response epitopes are set forth in SEQ ID NOs. 58-72.
Accordingly, RIC constructs targeting conserved epitopes of the NoV protruding domain (P-RIC), shell domain (S-RIC), T-cell epitopes (T-RIC), or containing tandem linked nonstructural protein 1 (NS1) from murine norovirus (N-RIC) are designed and produced. The unit of the antigen for P-RIC is formed from nine tandem-linked epitopes from the protruding domain of VP1 from GI.3, GII.4, and MNV. The unit of the antigen for P-RIC is formed from nine tandem-linked epitopes from the protruding domain of VP1 of GI.3, GII.4, and MNV. The unit of the antigen for S-RIC is formed from nine tandem-linked epitopes from the shell domain of VP1 of GI.3, GII.4, and MNV. The unit of the antigen for T-RIC is formed from fifteen tandem-linked epitopes that have been found to cause a T-cell mediated immune response against GI.3, GII.4, and MNV. The unit of the antigen for N-RIC is formed from tandem-linked epitopes from NS1 of GI.3, GII.4, and MNV. The plant expression vector for producing these RIC constructs are pBYKEAM-N12MHd, pBYKEAM-NPHd, pBYKEAM-NSHd, pBYKEAM-NTHd, pBYKEHM-CPHd, pBYKEHM-CSHd, and pBYKEHM-CTHd.
For each epitope, RIC constructs where the epitopes are linked to at the N-terminus or the C-terminus could be successfully expressed in plants (
The construction of a BeYDV plant expression vector for ZE3, as well as its fusion to the 6D8 C-terminus (pBYR11eM-h6D8ZE3), referred to here as construct “HLZe”) or N-terminus with epitope tag (pBYR11eMa-BAZE3-Hgp371) or without epitope tag (pBYR11eMa-BAZE3-H) was described in Diamos et al., 2019. A vector pBYKEMd2-6D8 expressing the full 6D8 mAb without ZE3 fusion (construct “HL”) was also described in Diamos et al., 2019. To create a vector expressing only the light chain of 6D8, pBYKEMd2-6D8 was digested with Xhol and the vector was self-ligated to yield pBYKEMd-6D8K. A vector expressing only the heavy chain of 6D8 (construct “H”) was created by digesting pBYKEMd2-6D8 with Sad and self-ligating the vector, to yield pBYKEMd-6D8H. The 6D8 epitope binding tag was added to pBYKEMd-6D8H by digesting pBYR11eMa-BAZE3-Hgp371 with BsaI-SacI and inserting the tag-containing fragment into pBYKEMd-6D8H digested with BsaI-SacI, yielding pBYKEMd-6D8Hgp371 (construct “HLe” when coexpressed with light chain). To remove the epitope tag from HLZe, pBYR11eM-h6D8ZE3 was digested with BamHI-SacI and ligated with a fragment containing ZE3 obtained via amplification with primers ZE3-Bam-F (5′-gcgggatccaagggcgtgtcatactcc, SEQ ID NO. 8) and ZE3-Sac-R (5′-acagagctcttaagtgctaccactcctgtg, SEQ ID NO. 9) and subsequent digestion with BamHI-SacI. The resulting vector, pBYKEMd-HZE3, was coinfiltrated with pBYKEMd-6D8K to produce construct “HLZ.” To produce ZE3 fused to the 6D8 N-terminus without light chain, pBYR11eMa-BAZE3-H was digested with Sad and the vector was self-ligated, yielding pBYKEMd-ZE3H (construct “ZH”). To introduce hexamer mutations, a region of the 6D8 heavy chain constant region was synthesized (Integrated DNA Technologies, Iowa, USA) containing the E345R, E430G, and S440Y mutations, then digested with BsaI-SacI and used to replace the BsaI-SacI region of 6D8 in pBYKEMd-ZE3H, yielding pBYKEMd-ZE3Hx (construct “ZHx”). RIC epitope tag mutant “a” was generated by annealing oligos 6D89-F (5′-ctagtgtttacaagctggacatatctgaggcataagagct, SEQ ID NO. 10) and 6D89-R (5′- cttatgcctcagatatgtccagcttgtaaaca, SEQ ID NO. 11) and ligating them into pBYR11eM-h6D8ZE3 digested SpeI-SacI; mutant “b” was generated by first amplifying mutant “a” with primers gpDISE-Sac-R: (5′-tttgagctcttactcagatatgtccagcttgtaaac, SEQ ID NO. 12) and 35S-F (5′aatcccactatccttcgc, SEQ ID NO. 13), then digesting the product with SpeI-SacI and ligating it into pBYR11eM-h6D8ZE3 digested with SpeI-SacI. Mutants “c” and “d” were created similarly to mutant “a” using overlapping oligos 6D87-F (5′-ctagttacaagctggacatatctgagtaagagct, SEQ ID NO. 14) and 6D87-R (5′-cttactcagatatgtccagcttgtaa, SEQ ID NO. 15) for “c” and 6D86-F (5′-ctagttacaagctggacatatcttaagagct, SEQ ID NO. 16) and 6D86-R (5′-cttaagatatgtccagcttgtaa, SEQ ID NO. 17).
In order to make a construct in which the variable heavy (VH) domain is linked to a variable light chain (VL) domain that, in turn, is directly fused to the constant region of the 6D8 antibody, the variable regions were first obtained through PCR amplification and end-tailoring of segments from pBYR11eM-h6D8ZE3. For the VH domain, the primers LIR-H3A (5′-aagcttgttgttgtgactccgag, SEQ ID NO. 18) and 6D8VH-Spe-R (5′-cggactagtagctgaagacactgtgac, SEQ ID NO. 19) were used. The VL region was obtained through PCR amplification of pBYR11eM-h6D8ZE3 with primers 35S-F (5′-aatcccactatccttcgc, SEQ ID NO. 13) and 6D8VK-Nhe-R (5′-cgtgctagccttgatctccactttggtc, SEQ ID NO. 20). In order to fuse VL region to the constant region of a human IgG antibody, a subclone was created by digesting the PCR fragment with XhoI-NheI and inserting it into a vector, pKS-HH-gp371, that contained the 6D8 heavy chain. This subclone was named pKS-VL. Next, pBYKEM-6D8K was digested with SbfI-SacI, the PCR product that amplified the variable heavy chain fragment was digested SbfI-SpeI, and the variable light chain subclone was digested SpeI-SacI. These fragments were assembled to create pBYKEMd2-VHLVK (HVL). Finally, this construct was used to create pBYKEMd2-HVLZe by a two-fragment ligation. The pBYKEMd2-VHLVK construct was digested with Bsal and Sad to obtain the vector fragment along with the variable regions of the heavy and light chains. To obtain the ZE3 antigen segment and the epitope tag, pBYR11eM-h6D8ZE3 was also digested Bsal-Sacl. The resulting construct, which was used to produce HVLZe, was named pBYKEMd-HVLZe.
The HVL, panel of constructs were created as follows. HVL-Hx, a construct containing a single-chain antibody with three point mutations in the Fc region in order to facilitate formation of single-chain hexamers, was created by a two-fragment ligation. The backbone was derived by a Bsal-SacI digest of pBYKEMd2-VHLVK and the insert containing the mutations for hexamer formation was derived from a BsaI-SacI digest of pBYKEMd-ZE3Hx. The final construct was named pBYKEMd2-HVL-Hx.
HVLZnt, a single-chain fusion with the ZE3 antigen on the C-terminus but no epitope tag, was created by ligating a BsaI-SacI digested backbone from pBYKEMd2-VHLVK to an insert derived from pBYKEMd-HZE3. This construct was named pBYKEMd2-HVLZnt
ZHVLnt, a single-chain RIC with the ZE3 antigen linked to the antibody N-terminus and no epitope tag, was created by the following ligation. The insert fragment containing the ZE3 antigen and the VH segment was amplified from pBYR11eMa-BAZE3-Hgp371 using the 35S-F and VH-BsaS-R primers. The VH-BsaS-R primer end-tailored the 5′ end to include a Bsal site that would result in a Spel overhang upon digestion with Bsal. This PCR fragment was digested Xhol-Bsal and ligated with a backbone XhoI-SpeI fragment obtained from pBYKEMd2-VHLVK. The final construct was named pBYKEMd2-ZHVLnt.
ZHVLe, a single-chain antibody RIC with an N-terminally fused ZE3 antigen and an epitope tag was created by a two-fragment ligation. pBYKEMd2-ZHVLnt was digested Bsal-Sacl to produce the vector segment containing the N-terminal ZE3 antigen and the HVL construct. The insert containing the 6D8 epitope tag was derived from a BsaI-SacI digest of pBYR11eMa-BAZE3-Hgp371. The final construct was named pBYKEMd2-ZHVLe.
ZHVLhx, a single-chain antibody with a N-terminally fused ZE3 antigen and point mutations to facilitate hexamer formation, was obtained by a two-fragment ligation. The vector fragment was obtained by a BsaI-SacI digest of pBYKEMd2-ZHVLnt and the insert fragment was derived from a BsaI-SacI digest of pBYKEMd2-HVL-Hx. The final construct was named pBYKEMd2- ZHVLhx.
The specific constructs described herein listed below:
Binary vectors were separately introduced into Agrobacterium tumefaciens EHA105 by electroporation. The resulting strains were verified by restriction digestion or PCR, grown overnight at 30° C., and used to infiltrate leaves of 5- to 6-week-old N. benthamiana maintained at 23-25° C. Briefly, the bacteria were pelleted by centrifugation for 5 min at 5,000 g and then resuspended in infiltration buffer (10 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5 and 10 mM MgSO4) to OD600=0.2, unless otherwise described. The resulting bacterial suspensions were injected by using a syringe without needle into leaves through a small puncture (Huang and Mason, 2004). To evaluate the effects of glycosylation, transgenic plants silenced for xylosyltransferase and fucosyltransferase were employed. Plant tissue was harvested at 5 DPI.
Crude protein was extracted by homogenizing agroinfiltrated leaf samples with 1:5 (w:v) ice cold extraction buffer (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 3 mM EDTA, 0.1% Triton X-100, 10 mg/mL sodium ascorbate, 0.3 mg/mL phenylmethylsulfonyl fluoride) using a Bullet Blender machine (Next Advance, Averill Park, N.Y.) following the manufacturer's instruction. Homogenized tissue was rotated at room temperature or 4° C. for 30 min. The crude plant extract was clarified by centrifugation at 13,000 g for 15 min at 4° C. and the supernatant was analyzed by SDS-PAGE or ELISA. Alternatively, to evaluate solubility of proteins in the original homogenate, the pellet was designated the insoluble fraction and treated with SDS sample buffer at 100° C. for 10 min before loading on SDS-PAGE.
IgG variants, including HVLZe, were purified by protein G affinity chromatography. Agroinfiltrated leaves were blended with 1:3 (w:v) ice cold extraction buffer (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 3 mM EDTA, 0.1% Triton X-100, 10 mg/mL sodium ascorbate, 0.3 mg/mL phenylmethylsulfonyl fluoride), stirred for 30 min at 4° C., and filtered through miracloth. To precipitate endogenous plant proteins, the pH was lowered to 4.5 with 1 M phosphoric acid for 5 min while stirring, then raised to 7.6 with 2 M tris base. Following centrifugation for 20 min at 16,000 g, the clarified extract was loaded onto a Pierce Protein G column (Thermo Fisher Scientific, Waltham, Mass., USA) following the manufacturer's instructions. Purified proteins were eluted with 100 mM glycine, pH 2.5, directly into collection tubes containing 1 M Tris-HCl pH 8.0 to neutralize the elution buffer. The HVL and ZHVLhx constructs were purified in a similar fashion; however, the acid precipitation step was skipped.
ZE3-His expressed from pBYe3R2K2Mc-BAZE3 was purified by metal affinity chromatography. Protein was extracted as described above, but without acid precipitation. The clarified extract was loaded onto a column containing TALON Metal Affinity Resin (BD Clontech, Mountain View, CA) according to the manufacturer's instructions. The column was washed with PBS and eluted with elution buffer (PBS, 150 mM imidazole, pH 7.4). Peak ZE3 elutions were pooled, dialyzed against PBS, and analyzed by SDS-PAGE and western blot.
Plant protein extracts or purified protein samples were mixed with SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.02% bromophenol blue) and separated on 4-15% stain-free polyacrylamide gels (Bio-Rad, Hercules, Calif., USA). For reducing conditions, 0.5M DTT was added, and the samples were boiled for 10 min prior to loading. Polyacrylamide gels were visualized and imaged under UV light, then transferred to a PVDF membrane. For IgG detection, the protein transferred membranes were blocked with 5% dry milk in PBST (PBS with 0.05% tween-20) overnight at 4° C. and probed with goat anti-human IgG-HRP (Sigma-Aldrich, St. Louis, Mo., USA diluted 1:5000 in 1% PBSTM). Bound antibody was detected with ECL reagent (Amersham, Little Chalfont, United Kingdom).
96-well high-binding polystyrene plates (Corning Inc, Corning, N.Y., USA) were coated with 15 μg/ml human complement C1q in PBS for 2 h at 37° C. The plate was washed 3 times with PBST, and then blocked with 5% dry milk in PBST for 15 minutes. After washing 3 times with PBST, purified human IgG (Southern Biotech, Birmingham, Al., USA) and purified IgG-ZE3 fusions were added at 0.1 mg/ml with 10-fold serial dilutions and incubated for 1.5h at 37° C. After washing 3 times with PBST, bound IgG was detected by incubating with 1:1000 polyclonal goat anti human IgG-HRP (Southern Biotech, Birmingham, Ala., USA) for lh at 37° C. The plate was washed 4 times with PBST, developed with TMB substrate (Thermo Fisher Scientific, Waltham, Mass., USA), stopped with 1M HCl, and the absorbance was read at 450 nm.
To test the ability of HVL to bind to the 6D8 epitope tag, 900 ng of purified dengue consensus envelope domain III tagged with 6D8 epitope were bound to 96-well high-binding polystyrene plates (Corning Inc, Corning, NY, USA). After a 1-hour incubation at 37° C., the plate was washed thrice with PBST and blocked with 5% dry milk in PBST for 30 minutes. Then, the plate was washed thrice with PB ST and various dilutions of either purified HLV or full-length 6D8 antibody were added to the plate. The plate was incubated at 37° C. for 1-hour, washed thrice with PBST and detected with HRP-conjugated mouse anti-human IgG (Fc only) (Southern Biotech, Birmingham, Ala., USA) antibody at a 1:2000 dilution. Then, the plate was thoroughly washed with PBST and developed with TMB substrate (Thermo Fisher Scientific, Waltham, Mass., USA). The absorbance was read at 450 nm.
Purified samples of each IgG fusion (100 μl) were loaded onto discontinuous sucrose gradients consisting of 350 μl layers of 5, 10, 15, 20, and 25% sucrose in PBS in a 2.0 ml microcentrifuge tube and centrifuged at 21,000 g for 16 h at 4° C. Fractions were collected and analyzed by SDS-PAGE followed by visualization on stain-free gels (Bio-Rad, Hercules, Calif., USA). The relative band intensity of each fraction was determined using ImageJ software, with the peak band arbitrarily assigned the value of 1.
All animals were handled in accordance to the Animal Welfare Act and Arizona State University IACUC. Female BALB/C mice, 6-8 weeks old, were immunized subcutaneously with purified IgG fusion variants. In all treatment groups, the total weight of antigen was set to deliver an equivalent 8 μg of ZE3. Doses were given on days 0 and 14. Serum collection was done as described in Santi et al., 2008 by submandibular bleed on days 0, 14, and 28.
Mouse antibody titers were measured by ELISA. Plant-expressed 6-His tagged ZE3 at 50 ng/well was bound to 96-well high-binding polystyrene plates (Corning Inc, Corning, NY, USA), and the plates were blocked with 5% nonfat dry milk in PBST. After washing the wells with PBST (PBS with 0.05% Tween 20), the mouse sera were diluted with 1% PBSTM (PBST with 1% nonfat dry milk) and incubated. Mouse antibodies were detected by incubation with polyclonal goat anti-mouse IgG-horseradish peroxidase conjugate (Sigma-Aldrich, St. Louis, Mo., USA). The plate was developed with TMB substrate (Thermo Fisher Scientific, Waltham, Mass., USA), stopped with 1M HCl, and the absorbance was read at 450 nm. Endpoint titers were taken as the reciprocal of the lowest dilution which produced an OD450 reading twice the background produced using PBS as the sample. IgG2a antibodies were measured from sera diluted 1:100 in 1% PBSTM and detected with IgG2a horseradish peroxidase conjugate (Santa Cruz Biotechnology, Dallas, Tex., USA).
This application claims the benefit of U.S. provisional patent application 62/980,012, filed Feb. 21, 2020 titled “IgG Variants for Induction of Neutralizing Immune Response Without Adjuvant,” the entirety of the disclosure of which is hereby incorporated by this reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/019090 | 2/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62980012 | Feb 2020 | US |
