BROADLY NEUTRALIZING ANTIBODY-TEMPLATED DIVALENT IMMUNOGEN VACCINES

[0001] This application claims priority to U.S. Provisional Application No. 63/430,144, filed Dec. 5, 2022, which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under NIH Training Grant #5K12HL143960-05 awarded by the National Institutes of Health (NIH). The United States Government, therefore, may have certain rights in the invention.

[0003] The present disclosure relates generally to divalent immunogens comprising a scaffold and two antigens or epitopes thereof, compositions, and methods of using the same. Such divalent immunogens are useful, for example, for stimulating an immune response.

[0004] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is IAMA_001_01WO_ST26.xml. The XML file is 113,944 bytes, was created on Dec. 4, 2023, and is being submitted electronically via USPTO Patent Center.

[0005] Vaccine design for rapidly-mutating viruses is challenging, and few successful vaccines have emerged in this area. Protective immune responses against such viruses often requires the elicitation of an immune response characterized by broadly neutralizing antibodies (bnAbs), which can neutralize phenotypically distinct variants of the virus. However, even for viruses for which bnAbs have been identified in patients (e.g., VRC01, a bnAb that binds the CD4 binding site of HIV gp120), eliciting expansion of the B cells that produce these bnAbs has proven difficult.

[0006] Expansion of a B cell requires binding of the B-cell receptor (BCR) expressed by that B cell to its cognate antigen. In the context of elicitation of an immune response, B cells expressing BCRs with a higher affinity for the antigen may out-compete those with lower affinity, leading to selective expansion of those with higher affinity. B cells expressing BCRs with a higher affinity for the antigen may survive the affinity maturation process while those with lower-affinity BCRs undergo apoptosis. If a bnAb germline antibody (and its corresponding BCR) has low affinity for its cognate antigen (e.g., VRC01 germline), B cells that express it may not expand sufficiently to generate protection against the virus. Thus, germline B cells must be activated by suitable immunogens to drive the evolution of their receptors toward the bnAb affinity and breadth. In the context of prime+boost vaccine regimens, germline B cells may need to evolve in a preferred direction through multiple bottlenecks in order to generate the preferred BCR (e.g., VRC01-class BCRs). New approaches are needed to selectively elicit expansion of such B cells.

[0007] In various embodiments, the present disclosure provides divalent immunogens comprising a scaffold and two antigens or epitopes thereof, wherein each antigen or epitope thereof is covalently linked to the scaffold. Also disclosed herein are multimers including these immunogens, pharmaceutical compositions including these immunogens, and polynucleotides encoding at least one antigen and/or epitope of the divalent immunogen, and/or the scaffold or a portion thereof. Also disclosed are methods of identifying antibodies for divalent immunogens described herein, and methods of use of divalent immunogens disclosed herein.

[0008] In one embodiment, the disclosure provides a divalent immunogen comprising: a scaffold, and two antigens or epitopes thereof; wherein each antigen or epitope thereof is covalently linked to the scaffold. In some embodiments, only a fraction of antigen-reactive B-cell receptors are able to bind to both antigens or epitopes thereof simultaneously and with 1:1 stoichiometry.

[0009] In some embodiments, the scaffold is derived from an antibody. In other embodiments, the scaffold is derived from a dimeric protein or a multimeric protein. In still other embodiments, the scaffold is derived from a monomeric protein. In some embodiments, each antigen or epitope thereof is expressed as a fusion polypeptide comprising at least a part or fragment of the scaffold. In some embodiments, the scaffold is a nucleic acid nanostructure.

[0010] In some embodiments, the scaffold comprises an antibody or fragment or variant thereof. In some embodiments, the scaffold comprises two Fab fragments or variants thereof. In some embodiments, the covalent linkage of the antigen or epitope thereof to the scaffold is a disulfide bond. In other embodiments, the covalent linkage is a chemical crosslinker, a protein crosslinker, or a nucleic acid crosslinker.

[0011] In some embodiments, each Fab fragment comprises a first engineered Fab mutation that enables formation of the disulfide bond between the Fab fragment and the antigen or epitope thereof. In some embodiments, each Fab fragment comprises a second engineered Fab mutation that enables Fab-Fab crosslinking, wherein the second engineered Fab mutation is in the light chain of each of the two Fab fragments. In some embodiments, each Fab fragment comprises a second engineered Fab mutation that enables Fab-Fab crosslinking, wherein the second engineered Fab mutation is in the heavy chain of each of the two Fab fragments. In some embodiments, the second engineered Fab mutation places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one antibody.

[0012] In some embodiments, the divalent immunogen comprises an engineered dimerization interface comprising C-terminal extension of the Fab heavy and/or light chains, optionally wherein the engineered dimerization interface places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one BCR and/or antibody.

[0013] In some embodiments, the divalent immunogen comprises a linker between the two antigens or epitopes thereof, optionally wherein the linker places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one BCR and/or antibody.

[0014] In some embodiments, the first antigen or epitope thereof is at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to the second antigen or epitope thereof. In some embodiments, the first antigen or epitope thereof is 100% identical to the second antigen or epitope thereof.

[0015] In some embodiments, each antigen or epitope thereof comprises a first engineered antigen mutation or a first engineered epitope mutation that enables formation of a disulfide bond between the scaffold and the antigen or epitope thereof.

[0016] In some embodiments, the divalent immunogen additionally comprises an Fc domain. In some embodiments, the Fc is a mouse Fc, optionally a mouse IgG2a Fc. In some embodiments, the Fc is a human Fc, optionally a human IgG1 Fc.

[0017] In some embodiments, the divalent immunogen comprises an engineered disulfide bond between the Fab and the Fc, optionally wherein the engineered disulfide bond places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one BCR and/or antibody. In some embodiments, the divalent immunogen comprises an engineered loop extension in the Fc, optionally wherein the engineered loop extension places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one BCR and/or antibody.

[0018] In some embodiments, all or part of the antibody is humanized. In some embodiments, the Fc comprises amino acid substitutions corresponding to L234A/L235A/P329G in SEQ ID NO 1.

[0019] In some embodiments, the antigen is HIV gp120, or a fragment or variant. In some embodiments, the epitope is the CD4 binding site of HIV gp120. In some embodiments, the sequence of the HIV gp120 antigen comprises a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99%, identical to SEQ ID NO: 13. In some embodiments, the sequence of the HIV gp120 antigen comprises SEQ ID NO: 13. In some embodiments, the sequence of the HIV gp120 antigen comprises SEQ ID NO: 15.

[0020] In some embodiments, the first engineered antigen mutation of the HIV gp120 antigen, or fragment or variant thereof, corresponds to 1423C of HIV gp120. In some embodiments, at least one of the two antigens or epitopes thereof does not comprise the N276 glycan.

[0021] In some embodiments, the Fab domains are derived from a co-receptor binding site antibody. In some embodiments, the co-receptor binding site antibody is 48d. In some embodiments, the sequence of the Fab heavy chain comprises a sequence at least 80% identical to SEQ ID NO: 1. In some embodiments, the sequence of the Fab light chain comprises a sequence at least 80% identical to SEQ ID NO: 3.

[0022] In some embodiments, the first engineered Fab mutation corresponds to D56 of the 48d heavy chain. In some embodiments, the second engineered Fab mutation corresponds to K126C of the 48d light chain. In some embodiments, the second engineered Fab mutation comprises addition of at least one amino acid between the residues corresponding to amino acids 119 to 132 of the 48d light chain, optionally wherein the second engineered mutation additionally comprises substitution of at least one of the residues corresponding to amino acids 119 to 132 of the 48d light chain. In some embodiments, the sequence of the Fab light chain comprises any one of SEQ ID NOs: 5, 7, 9, or 11.

[0023] In some embodiments, the divalent immunogen provides precise positioning of two antigens or epitopes thereof such that certain BCRs are preferably bound to both antigens simultaneously.

[0024] In some embodiments, the two Fab domains of the bound BCR assume an angle of about 20 degrees to about 150 degrees. In some embodiments, the two Fab domains of the bound BCR assume an angle of about 90 degrees to about 140 degrees. In some embodiments, the two Fab domains of the bound BCR assume an angle of about 120 degrees.

[0025] In some embodiments, the divalent immunogen provides an antigenic spacing of about 3 to about 20 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 7 to about 19 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 13 to about 16 nm.

[0026] In some embodiments, the second engineered Fab mutation fixes the distance between the antigens or epitopes thereof.

[0027] In some embodiments, the second engineered Fab mutation modifies the interaction between the antigens or epitopes thereof and certain BCRs. In some embodiments, the second engineered Fab mutation enhances the binding avidity of certain BCRs and/or antibodies for the antigen or epitope thereof by at least 1.5-fold. In some embodiments, the second engineered Fab mutation enhances the binding avidity of certain BCRs and/or antibodies for the antigen or epitope thereof by at least 10-fold. In some embodiments, the second engineered Fab mutation enhances the binding avidity of certain BCRs and/or antibodies for the antigen or epitope thereof by at least 100-fold.

[0028] In some embodiments, the binding avidity of certain BCRs is at least 1.5-fold higher for the two antigens covalently linked to the scaffold compared to the same antigen unlinked to the scaffold. In some embodiments, the binding avidity of certain BCRs is at least 10-fold higher for the two antigens covalently linked to the scaffold compared to the same antigen unlinked to the scaffold. In some embodiments, the binding avidity of certain BCRs is at least 100-fold higher for the two antigens covalently linked to the scaffold compared to the same antigen unlinked to the scaffold.

[0029] In a further embodiment, the present disclosure provides a multimer of one or more of the divalent immunogens disclosed herein. In some embodiments, the monomers of the divalent immunogen are conjugated onto a nanoparticle. In some embodiments, the multimer is a dimer, a trimer, or a tetramer.

[0030] In a further embodiment, the present disclosure provides a pharmaceutical composition comprising any one or more of the divalent immunogens and/or multimers disclosed herein. In some embodiments, the pharmaceutical composition additionally comprises a pharmaceutically-acceptable solvent. In some embodiments, the pharmaceutical composition additionally comprises an adjuvant, optionally wherein the adjuvant is poly I:C.

[0031] In a further embodiment, the present disclosure provides polynucleotides encoding a scaffold and/or two antigens or epitopes thereof disclosed herein. In some embodiments, the polynucleotide encodes a scaffold or portion thereof, wherein the encoded scaffold provides precise positioning of two antigens or epitopes thereof such that only certain BCRs are preferably bound to both antigens simultaneously. In some embodiments, the polynucleotide encodes a Fab heavy chain disclosed herein. In some embodiments, the polynucleotide encodes a Fab light chain disclosed herein.

[0032] In a further embodiment, the present disclosure provides a cell comprising at least one polynucleotide disclosed herein.

[0033] In a further embodiment, the present disclosure provides a method of identifying antibodies with preferred characteristics for divalent immunogens, comprising: (a) providing a divalent immunogen comprising a known broadly neutralizing antibody and an antigen or epitope thereof; (b) administering the divalent immunogen to a subject, optionally a mouse; (c) isolating B cells from the subject; (d) screening the isolated B cells for ability to bind to the divalent immunogen in high avidity, bidentate fashion; and (e) sequencing the complementarity determining regions of B cells that bind as in (d).

[0034] In a further embodiment, the present disclosure provides a method of generating an immune response in a patient, comprising administering one or more divalent immunogens, multimers, and/or pharmaceutical compositions disclosed herein. In some embodiments, the immune response results in an increased number of B cells expressing a BCR that binds the divalent immunogen divalently with increased avidity. In some embodiments, the patient has previously been administered at least one dose of a vaccine, optionally wherein the dose was of a different vaccine. In some embodiments, the immune response results in an increased number of B cells expressing a VRC01-class BCR.

[0035] In a further embodiment, the present disclosure provides a method of forming a ring structure comprising a divalent immunogen disclosed herein and a BCR or antibody; wherein the divalent immunogen comprises a specific antigenic spacing that allows the BCR or antibody to bind with a Fab-Fab angle of about 20 degrees to about 150 degrees. In some embodiments, the Fab-Fab angle is about 90 degrees to about 140 degrees. In some embodiments, the Fab-Fab angle is about 120 degrees. In some embodiments, the ring structure is formed in a subject. In some embodiments, the subject is a human.

[0055] The present disclosure provides divalent immunogens comprising a scaffold and two antigens or epitopes thereof, wherein each antigen or epitope thereof is covalently linked to the scaffold. The present disclosure also provides multimers thereof, pharmaceutical compositions thereof, and polynucleotides encoding at least one antigen and/or epitope of the divalent immunogen, and/or the scaffold or a portion thereof. The present disclosure also provides methods of identifying antibodies for divalent immunogens described herein, and methods of use of divalent immunogens disclosed herein.

[0056] The following terms are used in the description herein and in the appended claims.

[0057] The singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0058] The term “about” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0059] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0060] The terms “administering” or “introducing”, as used herein, refer to delivery of a composition to a cell, to cells, tissues and/or organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro or ex vivo.

[0061] The term “polynucleotide” as referred to herein means single-stranded or double-stranded nucleic acid polymers. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2′,3′-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term “polynucleotide” specifically includes single and double stranded forms of DNA.

[0062] The terms “polypeptide,” “protein,” “peptide,” and “glycoprotein” are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences.

[0063] The present disclosure provides divalent immunogens comprising a scaffold and two antigens or epitopes thereof. In some embodiments, each antigen or epitope thereof is covalently linked to the scaffold. As used herein, the term “immunogen” means a molecule or molecular assembly comprising at least one antigen or epitope. In some embodiments, the divalent immunogen comprises one polypeptide, two polypeptides, three polypeptides, four polypeptides, five polypeptides, six polypeptides, seven polypeptides, or eight polypeptides. Non-limiting applications for immunogens include administration to a subject to elicit an immune response.

[0064] As used herein, the term “scaffold” refers to a molecule or molecular assembly suitable for spatially orienting two antigens or epitopes thereof. In some embodiments, the scaffold comprises one polypeptide, two polypeptides, three polypeptides, four polypeptides, five polypeptides, six polypeptides, seven polypeptides, or eight polypeptides. In some embodiments, each antigen or epitope thereof is covalently linked to the scaffold. Covalent linkage may be via any method known in the art. Non-limiting examples of covalent linkage are disulfide bonds and peptide bonds.

[0065] The divalent immunogens disclosed herein leverage the concept of avidity to enhance immune focusing (i.e., targeting specific epitopes for antibody response). The disclosed divalent immunogens provide the precise positioning of two antigens such that only certain B-cell receptors (BCRs), or the corresponding soluble antibodies, can bind to the antigens with both Fabs simultaneously, forming a ring dimer structure (shown in FIG. 1B). With significant rigidity of the divalent antigen and ideal binding geometry of targeted BCRs, the avidity effect is expected to be greatest. Optimal rigidity and binding geometry also minimize the entropic penalty for divalent binding—this is a significant factor especially when the monovalent affinity of the BCR (or antibody) is low.

[0066] In some embodiments, the scaffold is derived from an antibody. In some embodiments, the scaffold comprises an antibody or a fragment or variant thereof.

[0067] As is well known in the art, an antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one epitope recognition site, located in the variable region of the immunoglobulin molecule. Antibody fragments are encompassed by the present disclosure, such as Fab, F(ab′) 2, and Fv which include a heavy chain and light chain variable region and specifically bind a certain antigen. These antibody fragments retain the ability to selectively bind with the antigen.

[0068] These fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)₂, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′) 2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. (6) A dimer of a single chain antibody (scFV₂), defined as a dimer of a scFV. This has also been termed a “miniantibody.” Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).

[0069] In some embodiments, an antibody or antigen-binding fragments thereof, e.g., a Fab, scFv, VHH or sdAb, may have: a) a heavy chain having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the heavy chain of an antibody or antigen-binding fragments thereof described herein; and/or b) a light chain having an amino acid sequence that is at least 80% identical, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the light chain of an antibody or antigen-binding fragments thereof described herein. The amino acid sequences of illustrative antibodies and fragments thereof are set forth herein.

[0070] The antibody from which the scaffold is derived may be from any known class. Non-limiting examples include IgA (including subclasses IgAQ1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM.

[0071] In some embodiments, the scaffold comprises two Fab fragments or variants thereof. In some embodiments, each antigen or epitope is covalently linked to a Fab fragment. Non-limiting examples of covalent linkers are disulfide bonds, peptide bonds, chemical crosslinkers, protein crosslinkers, and nucleic acid crosslinkers.

[0072] In some embodiments, the Fab fragments are derived from a co-receptor binding site antibody. In some embodiments, the Fab fragments comprise a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the heavy chain sequence of a co-receptor antibody. In some embodiments, the Fab fragments comprise a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the light chain sequence of a co-receptor antibody.

[0073] In some embodiments, the co-receptor binding site antibody is 48d. In some embodiments, the Fab fragments comprise a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the heavy chain sequence of the 48d antibody. In some embodiments, the Fab fragments comprise a light chain sequence at least at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the light chain sequence of the 48d antibody. In some embodiments, the Fab fragments comprise a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the heavy chain sequence of SEQ ID NO: 1. In some embodiments, the Fab fragments comprise a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the light chain sequence of SEQ ID NO: 3. In some embodiments, the Fab fragments comprise a heavy chain sequence comprising SEQ ID NO: 1. In some embodiments, the Fab fragments comprise a light chain sequence comprising SEQ ID NO: 3. In some embodiments, the Fab fragments comprise a heavy chain sequence comprising SEQ ID NO: 1 and a light chain sequence comprising SEQ ID NO: 3.

[0074] In some embodiments, the Fab fragments comprise a light chain sequence comprising SEQ ID NO: 5. In some embodiments, the Fab fragments comprise a light chain sequence comprising any one of SEQ ID NOs: 7, 9, and 11. In some embodiments, the Fab fragments comprise a heavy chain sequence comprising SEQ ID NO: 1 and a light chain sequence comprising SEQ ID NO: 5. In some embodiments, the Fab fragments comprise a heavy chain sequence comprising SEQ ID NO: 1 and a light chain sequence comprising SEQ ID NO: 7. In some embodiments, the Fab fragments comprise a heavy chain sequence comprising SEQ ID NO: 1 and a light chain sequence comprising SEQ ID NO: 9. In some embodiments, the Fab fragments comprise a heavy chain sequence comprising SEQ ID NO: 1 and a light chain sequence comprising SEQ ID NO: 11.

[0075] In some embodiments, each Fab fragment comprises at least a first engineered Fab mutation. In some embodiments, the first engineered Fab mutation enables formation of the disulfide bond between the Fab fragment and the antigen or epitope thereof. Non-limiting examples of engineered mutations include addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of the Fab fragment. In some embodiments, the mutation comprises addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of the Fab heavy chain sequence. In other embodiments, the mutation comprises addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of the Fab light chain sequence. In some embodiments, the first engineered Fab mutation comprises a substitution corresponding to D56C of SEQ ID NO: 1.

[0076] In some embodiments, each Fab fragment comprises at least a second engineered Fab mutation that enables Fab-Fab crosslinking. In some embodiments, the second engineered Fab mutation is in the light chain of each of the two Fab fragments. In some embodiments, the second engineered Fab mutation is in the heavy chain of each of the two Fab fragments. In some embodiments, the mutation comprises addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of the Fab heavy chain sequence. In other embodiments, the mutation comprises addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of the Fab light chain sequence. In some embodiments, the second engineered Fab mutation comprises a substitution corresponding to K126C of SEQ ID NO: 3.

[0077] In some embodiments, the second engineered Fab mutation places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one antibody. In some embodiments, the second engineered Fab mutation comprises addition of at least one amino acid between the residues corresponding to amino acids 119 to 132 of SEQ ID NO: 3. In some embodiments, the second engineered mutation additionally comprises substitution of at least one of the residues corresponding to amino acids 119 to 132 of SEQ ID NO: 3.

[0078] In some embodiments wherein the scaffold comprises a Fab domain, the divalent immunogen comprising the Fab additionally comprises an Fc domain. The Fc region may be derived from any of a variety of different Fcs, including but not limited to, a wild-type or modified IgG1, IgG2, IgG3, IgG4 or other isotype, e.g., wild-type or modified human or murine IgG1, human or murine IgG2, human or murine IgG3, human IgG4, human IgG4Pro (comprising a mutation in core hinge region that prevents the formation of IgG4 half molecules), human or murine IgA, human or murine IgE, human or murine IgM, or human or murine IgM. In some embodiments, the Fc domain is derived from a human IgG1. In some embodiments, the Fc domain is derived from a murine IgG2a. In some embodiments, all or part of the Fc is humanized.

[0079] A “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A “humanized antibody” is an antibody comprising a humanized light chain and/or a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (for example, see U.S. Pat. No. 5,585,089).

[0080] In some embodiments, the divalent immunogen comprises an engineered dimerization interface comprising C-terminal extension of the Fab heavy and/or light chains. In some embodiments, the divalent immunogen comprises an engineered dimerization interface comprising C-terminal extension of the Fc heavy and/or light chains. The C-terminal extension may comprise addition or insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or least 20 amino acids to the Fab and/or Fc heavy and/or light chains. In some embodiments, the engineered dimerization interface places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one BCR and/or antibody.

[0081] In some embodiments, the Fc comprises amino acid substitutions corresponding to L234A, L235A, P329G (LALA-PG) in SEQ ID NO: 1 and/or as described in Lo et al. J. Biol. Chem. 2017; 292, p. 3900-3908. The LALA-PG variant has been shown to eliminate complement binding and fixation as well as Fc-g dependent antibody-dependent cell-mediated cytotoxicity (ADCC) in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an “effectorless” antibody framework scaffold between mice and primates.

[0082] The present disclosure also provides divalent immunogens wherein the divalent immunogen comprises an engineered disulfide bond between the Fab and the Fc, optionally wherein the engineered disulfide bond places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one antibody.

[0083] Divalent immunogens that position the antigens ideally for engagement by BCRs may also be designed by creating a covalent bond between the Fabs and the Fc (e.g., an engineered disulfide or chemical crosslinking, as in FIG. 10A). Disulfide formation could be accomplished by, for example, point mutations within Fab and Fc to cysteines, and/or loop extension in the Fab and/or Fc and incorporation of cysteines. Chemical crosslinking could be accomplished by e.g. cysteine point mutation/dimaleimide crosslinking. Divalent immunogens that position the antigens ideally for engagement by BCRs may also be designed by engineering a rigid antibody structure by adding bulky loop extensions (e.g., as in FIG. 10B), by adding a loop extension or extension of the C-termini of the heavy and/or light chains to come together and form a stable Fab dimer interface (e.g., as in FIG. 11A), or by adding a linker connecting the two antigens that holds them at the ideal orientation (e.g., as in FIG. 11B).

[0084] In some embodiments, the divalent immunogen comprises a linker between the two antigens or epitopes thereof. Non-limiting examples of the linker are a peptide linker, a nucleic acid linker, concatamerization of the antigens in a single gene, connection by chemical crosslinking, and connection using exogenous proteins. In some embodiments, the linker places the antigens or epitopes thereof in a more favorable orientation for divalent binding of at least one BCR and/or antibody.

[0085] In some embodiments, the scaffold is derived from a dimeric protein. In other embodiments, the scaffold is derived from a multimeric protein. In still other embodiments, the scaffold is derived from a monomeric protein. Non-limiting examples of divalent immunogens incorporating a scaffold based on a dimeric or multimeric protein are shown in FIGS. 16 and 17, and in SEQ ID NOs: 71 and 72.

[0086] In some embodiments, the scaffold comprises a partially de novo engineered protein scaffold. Scaffolds comprising a partially de novo engineered protein scaffold may be based on a monomeric, dimeric, or multimeric protein.

[0087] In other embodiments, the scaffold comprises a completely de novo engineered protein scaffold. Scaffolds comprising a completely de novo engineered protein scaffold may be based on a monomeric, dimeric, or multimeric protein.

[0088] In other embodiments, the scaffold comprises a nucleic acid nanostructure. Non-limiting examples of a nucleic acid nanostructure include a DNA nanostructure (Seeman and Sleiman. Nat Rev Mater. 2018. 3, 17068).

[0089] Non-limiting examples of sequences that such a divalent immunogen comprising a scaffold the comprises a partially or fully de novo engineered protein scaffold include SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 63, 65, 67, 69, and 71.

[0090] In other embodiments, the divalent immunogen comprises a fusion protein of an antibody light chain (e.g., the 48d antibody light chain) and one antigen (e.g., gp120) expressed as a single polypeptide. In this embodiment, the C-terminus of the antigen is fused with the N-terminus of the light chain. The non-fused heavy chain is co-expressed. Two copies of the fusion protein and two copies of the non-fused heavy chain together form a divalent immunogen. A non-limiting example of a sequence that such a divalent immunogen comprising a fusion protein may comprise is SEQ ID NO: 61.

[0091] In other embodiments, the divalent immunogen comprises a fusion protein of an antibody heavy chain (e.g., the 48d antibody heavy chain) and one antigen (e.g., gp120) expressed as a single polypeptide. In this embodiment, the C-terminus of the antigen is fused with the N-terminus of the heavy chain. The non-fused light chain is co-expressed. Two copies of the fusion protein and two copies of the non-fused light chain together form a divalent immunogen. A non-limiting example of a sequence that such a divalent immunogen comprising a fusion protein may comprise is SEQ ID NO: 17.

[0092] Non-limiting examples of sequences that the divalent immunogen scaffold may comprise are shown in Table 1 and SEQ ID NOs: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 61, 63, 65, 67, 69, and 71.

TABLE 1
Non-limiting examples of polypeptides comprised by divalent immunogen
scaffolds
	SEQ
	ID
ID	NO.	Sequence
48d	1	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
antibody		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
HC		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPNAAGGPSVFIFPPKIKDVLMI
		SLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI
		QHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ
		VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKN
		WVERNSYSCSVVHEGLHNHHTTKSFSRTPG
48d	3	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
antibody LC		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNRGEC
48d	5	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
antibody LC		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
K126C		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLCSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNRGEC
48d	7	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
antibody LC		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
Loop1		SFFTFGGGTKVEIKRTVAAPSVFIFPPNDEQLLPCAGPSNASVVCLLNNFYPREAKV
		QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
		LSSPVTKSFNRGEC
48d	9	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
antibody LC		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
Loop2		SFFTFGGGTKVEIKRTVAAPSVFIFPPAPPEGCGSPTRTVVCLLNNFYPREAKVQW
		KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
		PVTKSFNRGEC
48d	11	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
antibody LC		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
Loop3		SFFTFGGGTKVEIKRTVAAPSVFIFRAEEGCGGNTVYLVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNRGEC
gp120.Core.	17	MPMGSLQPLATLYLLGMLVASVLADIRQAHCNVSKATWNETLGKVVKQLRKHF
48D + 48D.		GNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSN
Heavy_		DSITLPCRIKQCINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFR
Fusion		PGGGDMRDNWRSELYKYKVVKIEGGVWKDAETTLFCASDAKAYETEKHNVWA
		THACVPTDPNPQEIHLEGVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTG
		GSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPV
		VSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPGNGGGGGSGGG
		SGGGSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMYWVRQAPGKGLEWM
		GLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSEDTAVYYCAADPWEL
		NAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
		SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
		KKVEPKSCDKTHTCPPCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED
		DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV
		NNKDLGAPIERTISKPKGSVRAPQVYVIPPPEEEMTKKQVTLTCMVTDFMPEDIY
		VEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEG
		LHNHHTTKSFSRTPG
RFD1_Heavy	19	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVTANTEVTPESVRAALRELFGDEELLAWAAELLRA
RFD1_Light	21	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTVSYEKLGPGLWIYQTIQDGKLLSISTNAKPSTPLEEKVRAVAIREKLIAENAA
RFD1_Light.	23	MPMGSLQPLATLYLLGMLVASVLASAITQACPKVSFEPIPIHYCAPAGFAILKCKD
gpFusion:		KKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILV
		QFNTPVQINCTRPGNGGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFA
		NSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRI
		KQCINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMR
		DNWRSELYKYKVVKIEGGVWKDAETTLFCASDAKAYETEKHNVWATHACVPTD
		PNPQEIHLEGVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTGGGSGGGSG
		GGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAAS
		TLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQANSFFTFGGGTKVEIKRTV
		AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
		QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTVSYEKLGPGLWIYQ
		TIQDGKLLSISTNAKPSTPLEEKVRAVAIREKLIAENAA
RFD2_Heavy	25	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVSTPVEAVLPPEALAHLRAFLALLPLEAALAVAHAVRQ
		ALIAVQ
RFD2_Light	27	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTARILRVNDVVFTSTQTETSGEWTVNSTPLDQLLLDIYLEAQAA
RFD3_Heavy	29	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVEPKNSVTLTSTAADRELEEALRKLAPEAKFVLVGTLP
		DGSTLVIVFVS
RFD3_Light	31	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNKYPGVVVTETVDEATGTTTITLNTKNYKAVRLAIKIAEALHKLRPGSTTV
		AVIS
RFD4_Heavy	33	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVIITFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVPTNKVVTVTIEVKEEELPEDVSVRIRMEIDISEKTGKV
		EMRIEVSKS
RFD4_Light	35	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTVTIHRFKPNNVVTIEKLPSGKIKVTLTAENVEEALVNAYRLALELAEE
RFD5_Heavy	37	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVEPKNWVKATVKVEDGVVEAEFDSGGSKNIIEGLANS
		MKLLAEVALELLS
RFD5_Light	39	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNRLSSGTWATIEKKEDPGEMNVTANVANVISAKIKVEFK
RFD6_Heavy	41	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVEPKEVLNRVTVKVPAELTEAEIAMLKTLLKALGEAIA
		KAAK
RFD6_Light	43	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNKETGLETDVDPKDADTPEYKRVTVYTPLTAEDVELFKKLAEKLAEAIYE
		AAKER
RFD7_Heavy	45	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVEPKNGFSLSGSPEEVVAQALKHHTGDEKVAKMGVAL
		VKELLA
RFD7_Light	47	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNRYPSGVIVYRTTSNGSSSYGIDANLKTLEALLEFIEAVVEIYKER
48D_H.C56D.	49	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
MouseIgG2a.		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
Kdel.		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
Spy:		LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPNAAGGPSVFIFPPKIKDVLMI
		SLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI
		QHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ
		VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKN
		WVERNSYSCSVVHEGLHNHHTTKSFSRTPGGSGGSGGSGRGVPHIVMVDAYKRY
		K
48D_H.	51	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
ShortUpperHinge.		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
MouseIgG2a.		DTAVYYCAADPWELNAFNVWGQGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAA
Kde1.Spy		LGCLVKDYFPEPVTVSWNSGALTSGVIITFPAVLQSSGLYSLSSVVTVPSSSLGTQT
		YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPNAAGGPSVFIFPPKIKDVLMI
		SLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI
		QHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ
		VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKN
		WVERNSYSCSVVHEGLHNHHTTKSFSRTPGGSGGSGGSGRGVPHIVMVDAYKRY
		K
gp120.Core.	61	MPMGSLQPLATLYLLGMLVASVLASAITQACPKVSFEPIPIHYCAPAGFAILKCKD
48D + 48D.		KKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILV
Light_Fusion		QFNTPVQINCTRPGNGGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFA
		NSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRI
		KQCINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMR
		DNWRSELYKYKVVKIEGGVWKDAETTLFCASDAKAYETEKHNVWATHACVPTD
		PNPQEIHLEGVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTGGGSGGGSG
		GGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAAS
		TLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQANSFFTFGGGTKVEIKRTV
		AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
		QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
RFD2_v2_	63	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
Heavy		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSGAAVTGPRVFPLSPAGCSTTSPTTL
		LGCLVDDYYPLPVTVTWNGGALTSGVTTFPAVLRPDGLYSQVSVISVPSSSCGTE
		TYTCNVTHPASNTTVSQLVPCPRQLELPAEALAHLRAFLALLPPAAALAVAHAVL
		QALAAAL
RFD2_v2_Light	65	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKILIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVVAPSVSIFPPSPEELASGRARVVCVASSFYPESLEVTWLV
		DGEPVSGRSFTSTSAKNPADNTYSLVSTLDLSTEEYLAHRRFECRVTHSGLSSPVT
		ASILRVGDTLFTSVSSSDDGVFTVTGSPLMQLALDLYLEAQAA
RFD3_v2_	67	MGWSCIILFLVATATGVHSEVQLVQSGAEVKKPGATVKISCKASGYTFSDFYMY
Heavy		WVRQAPGKGLEWMGLIDPEDACTMYAEKFRGRVTITADTSTDTGYLELSSLRSE
		DTAVYYCAADPWELNAFNVWGQGTLVSVSGAPVTGPEVFPLPPMSVGSGKPSAT
		LGCLVKNFYPLPVEVTWNGGAVTEGVYVFPPVLRPDGRWSLVSVLRVPESTLGT
		ETYVCNVRHPPANVTVSKEVKALPGVTLTQTPEDAALEAKLRKACPEAEAIIVGT
		LPNGATLVLVFVP
RFD3_v2_Light	69	MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWY
		QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQAN
		SFFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
		VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
		VTKSFNKYPGVVVTETVDEATGTTTITLNTKNYKAVRLAIKIAEALHKLRPGSTTV
		AVIS
gp120-BAR	70	LEAKAKALKAALDAAAAAAAAAAAAYLALLAATEAAVAAALAKIPDPATRAAR
		AALAAAFAAALRGFATDLIAGGYAGLKERAEEIVELEKERAALAARRADLAAQA
		AALAALEAEGTRLQRELRGYLAAIKGMQEASMKLTESLHEVYEPDWYGREDVK
		MVGEKCDVLWEDFHQKLVDESLLTLDTYLGQFPALRARLAALAAQAAALEAAL
		QALIDLLQKKPYGDAETTLFCASDAKAYETEKHNVWATHACVPAVPRFETPALP
		VTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCNCNTSAITQACPKVSFE
		PIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEE
		EVMIRSENITNNAKNILVQFNTPVQINCTRPNDIIGDIRQAHCNVSKATWNETLGK
		VVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNND
		SITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRP
		GGGDMRDNWRSELYKYKVVKPLSEDPELKAARAAYAAARAAYDAAYDALAAE
		LPALLSRRVGFYVNTFKNVSSLEAKFHKEIAVLCHKLYEVMTKLGDQHA
1gh7_Fusion_	71	EETIPLQTLRCYNDYTSHITCRWADTQDAQRLVNVTLIRRVNEDLLEPVSCDLSDD
008		MPWSACPHPRCVPRRCVIPCQSFVVTDVDYFSFQPDRPLGTRLTVTLTQHVQPPEP
		RDLQISTDQDHFLLTWSVALGSPQSHWLSPGDLEFEVVYKRLQDSWEDAAILLSN
		TSQATLGPEHLMPSSTYVARVRTRLAPGSRLSGRPSKWSPEVCWDSQPGDEAQPQ
		NLECFFDGAAVLSCSWEVRKEVASSVSFGLFYKPSPREEECSPVLREGLGSLHTRH
		HCQIPVPDPATHGQYIVSVQPRRAEKHIKSSVNPTYYETFPNYQVIYLPTTTTDPSN
		KEPLGNTVTPAPVGTVTTYTPDGTRSSAITQACPKVTFEPIPIHYCAPAGFAILKCK
		DEEFNGTGLCKNVSTVQCTHGIKPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIV
		QLTKPVLINCARPSGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTTIIFNEPSG
		GDLEITTHSFNCGGEFFYCNTSDLFNSTWNDNITLPCRIKQIINMWQRVGQAMYAP
		PIQGVIRCQSNITGILLTRDGGNTRTNETFRPGGGDMRDNWRSELYKYKVVKIE

[0093] The divalent scaffolds disclosed herein may be especially well suited for presentation of antigens from diseases or conditions for which eliciting a protective or therapeutic immune response has proven challenging. Non-limiting examples of such diseases include viruses for which elicitation of an immune response comprising a preferred neutralizing antibody (e.g., a broadly neutralizing antibody) would be expected to be protective or therapeutic. Additional non-limiting examples of such diseases include rapidly-mutating viruses. Non-limiting examples of such viruses include human immunodeficiency virus (HIV), influenza A, influenza B, severe acute respiratory syndrome coronaviruses (SARS-COV-1 and SARS-CoV-2), sarbecoviruses, hepatitis C virus, respiratory syncytial virus, dengue virus, Ebola virus, and norovirus.

[0094] Suitable antigens for presentation as part of the divalent immunogens described herein may include those exposed on the surface of a virus. Suitable antigens may also include any antigens recognized by neutralizing antibodies which, if elicited, would be expected to contribute to a protective or therapeutic immune response. In some embodiments, the antigen may be a viral spike protein, envelope protein, membrane protein, or fusion protein. Non-limiting examples of antigens for presentation by the disclosed divalent immunogens include HIV envelope protein, Influenza A Hemagglutinin, Influenza B Hemagglutinin, SARS-COV-2 spike protein, hepatitis C virus E2 protein, respiratory syncytial virus fusion (F) protein, and Ebola virus glycoprotein. In some embodiments, the antigen comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 13, 15, and 54-60. In some embodiments, the antigen comprises any one of SEQ ID NOs: 13, 15, and SEQ ID NOs: 54-60.

[0095] In some embodiments, the antigen comprises HIV gp120, or HIV gp120 Core, or a fragment or variant thereof. In some embodiments, the antigen comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the antigen comprises SEQ ID NO: 13. In some embodiments, the antigen comprises SEQ ID NO: 15.

[0096] In some embodiments, the epitope is the CD4 binding site of HIV gp120, or a fragment or variant thereof. In some embodiments, the epitope comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to residues 123-124, 275-282, 365-371, 427-432, and 455-476 of SEQ ID NO: 53. In some embodiments, the epitope comprises residues corresponding to amino acids 123-124, 275-282, 365-371, 427-432, and 455-476 of SEQ ID NO: 53.

[0097] In some embodiments, presentation of non-identical but genetically related antigens may be advantageous. In a non-limiting example, presentation of non-identical but genetically related antigens may be advantageous for the divalent, high-avidity binding of BCRs that have broad reactivity. In this setting, the inclusion of two antigens from distantly related isolates of the same virus, each bound to the scaffold, would result in high avidity, divalent binding by BCRs that are able to bind to conserved features between the strains, which can promote broad reactivity.

[0098] In some embodiments, the first antigen or epitope thereof is 100% identical to the second antigen or epitope thereof. In some embodiments, the first antigen or epitope thereof is less than 100% identical to the second antigen or epitope thereof. For example, the first antigen or epitope thereof may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the second antigen or epitope thereof.

[0099] Percentage “sequence identity” of one polypeptide with respect to another polypeptide refers to the percentage of amino acids of the first polypeptide that are the same as the second polypeptide, when comparing the two aligned sequences. Sequence similarity can be determined via a number of different methods. For example, sequence similarity or identity may be determined using the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch J Mol. Biol. 48, 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al. Nucl. Acid Res. 12, 387-395 (1984), or by inspection.

[0100] Another suitable algorithm is the BLAST algorithm, described in Altschul et al. J. Mol. Biol. 215, 403-410, (1990) and Karlin et al. Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al. Methods in Enzymology, 266, 460-480 (1996); blast.wust1/edu/blast/README.html. WU-BLAST-2 uses several search parameters, which are optionally set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. Further, an additional useful algorithm is gapped BLAST as reported by Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise indicated, percent identity is determined herein using the algorithm available at the internet address: blast.ncbi.nlm.nih.gov/Blast.cgi.

[0101] In some embodiments, each antigen or epitope thereof comprises a first engineered antigen mutation or a first engineered epitope mutation. In some embodiments, the first engineered antigen mutation or first engineered epitope mutation enables formation of a disulfide bond between the scaffold and the antigen or epitope thereof. Non-limiting examples of engineered mutations include addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of the Fab fragment. In some embodiments, the first engineered antigen mutation corresponds to 1423C of SEQ ID NO: 53.

[0102] Incorporation of heterodimeric antigens into the divalent immunogen may be achieved via a number of mechanisms. A non-limiting example of such a mechanism is through use of bivalent antibody technology, where asymmetry is engineered into the complex (e.g., in the Fc region or any other scaffold region) such that only heterodimers (e.g., of an antigen-Fab fusion protein, for example) can stably form. Other non-limiting examples of such a mechanism are to attach a different affinity tag to each half of the antibody and then purify for both tags, or to engineer the heterodimeric antigen on a single polypeptide chain.

[0103] In some embodiments, the first and second antigen of the divalent immunogen differ at least in the presence of specific glycans. For example, a glycan at a specific position (e.g., a specific amino acid) can be present on the first antigen and absent on the second antigen, or present on the second antigen and absent on the first antigen. A glycan can also be present on both the first and second antigens or absent on both the first and second antigens. A non-limiting example of such a glycan is the glycan at position 276 of HIV antigen gp120. In some embodiments, the first gp 120 antigen comprises a glycan at position 276 and the second gp120 antigen does not comprise a glycan at position 276. In some embodiments, the first gp120 antigen does not comprise a glycan at position 276 and the second gp120 antigen comprises a glycan at position 276. It is well-established that a major obstacle for VRC01 lineage B-cells is overcoming the steric restriction caused by a glycan at this position. Immunogens that uniformly incorporate this glycan across all constituting gp120 antigens may offer an insurmountable challenge to the available BCR repertoire of VRC01-lineage B-cells. Divalent antigens that that differ in glycan occupancy at position 276 may provide an intermediate level of avidity such that B-cells whose receptors gain mutations marginally effective at accommodating the glycan at that position are positively selected.

[0104] Non-limiting examples of sequences that the divalent immunogen antigens may comprise are shown in Table 2 and SEQ ID NOs: 13-16 and SEQ ID NOs: 53-59.

TABLE 2
Non-limiting examples of polypeptides comprised by divalent immunogen
antigens
gp120.	SEQ ID
Core	NO.	Sequence
gp120.	13	MPMGSLQPLATLYLLGMLVASVLAVWKDAETTLFCASDAKAYETEKHNVWATHAC
Core		VPTDPNPQEIHLEGVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTGGSAITQA
		CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNG
		SLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPGNGGDIRQAHCNVSKATWNETL
		GKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTS
		VQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGCST
		NSTTETFRPGGGDMRDNWRSELYKYKVVKIE
gp120.	14	ATGCCCATGGGCTCCCTGCAGCCGTTAGCAACTCTGTATCTGCTGGGGATGCTGGT
Core.		AGCCAGCGTGCTGGCAGTGTGGAAAGATGCTGAAACCACATTGTTTTGTGCGAGC
48D		GATGCCAAAGCATACGAGACGGAAAAGCACAATGTCTGGGCCACTCATGCATGC
		GTGCCCACAGACCCAAACCCCCAGGAGATCCACCTGGAAGGAGTGACCGAGGAA
		TTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACAGACATCATTAGCC
		TGTGGGATCAGTCCCTGAAGCCCTGCGTCAAACTGACTGGCGGCTCCGCCATTAC
		CCAGGCTTGTCCTAAAGTGTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAG
		CTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTG
		CCCTTCCGTGTCTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACAC
		AGCTGCTGCTGAATGGAAGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGA
		ACATTACCAACAATGCCAAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGAT
		TAATTGCACAAGACCCGGAAATGGAGGAGATATCAGACAGGCTCACTGTAATGTG
		AGTAAGGCAACCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAA
		CATTTCGGGAATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGG
		AGGTCACTACCCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCT
		GGCCTGTTTAATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAA
		CTGGGTCAAACGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATAT
		GTGGCAGCGGATAGGTCAGGCAATGTATGCTCCCCCAATCCAGGGAGTTATTCGG
		TGCGTCTCTAATATCACGGGACTTATCTTGACTCGCGACGGCTGCAGCACGAACA
		GCACAACAGAAACATTCCGGCCTGGGGGGGGAGACATGCGGGATAACTGGCGGA
		GTGAGCTGTACAAGTATAAAGTTGTTAAGATCGAATGA
gp120.	15	MPMGSLQPLATLYLLGMLVASVLAVWKDAETTLFCASDAKAYETEKHNVWATHAC
Core.		VPTDPNPQEIHLEGVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTGGSAITQA
48D		CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNG
		SLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPGNGGDIRQAHCNVSKATWNETL
		GKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTS
		VQGSNSTGSNDSITLPCRIKQCINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGS
		TNSTTETFRPGGGDMRDNWRSELYKYKVVKIE
gp120	16	ATGCCCATGGGCTCCCTGCAGCCGTTAGCAACTCTGTATCTGCTGGGGATGCTGGT
from		AGCCAGCGTGCTGGCAGTGTGGAAAGATGCTGAAACCACATTGTTTTGTGCGAGC
HXBc2		GATGCCAAAGCATACGAGACGGAAAAGCACAATGTCTGGGCCACTCATGCATGC
SEQ		GTGCCCACAGACCCAAACCCCCAGGAGATCCACCTGGAAGGAGTGACCGAGGAA
		TTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACAGACATCATTAGCC
		TGTGGGATCAGTCCCTGAAGCCCTGCGTCAAACTGACTGGCGGCTCCGCCATTAC
		CCAGGCTTGTCCTAAAGTGTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAG
		CTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTG
		CCCTTCCGTGTCTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACAC
		AGCTGCTGCTGAATGGAAGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGA
		ACATTACCAACAATGCCAAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGAT
		TAATTGCACAAGACCCGGAAATGGAGGAGATATCAGACAGGCTCACTGTAATGTG
		AGTAAGGCAACCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAA
		CATTTCGGGAATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGG
		AGGTCACTACCCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCT
		GGCCTGTTTAATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAA
		CTGGGTCAAACGATAGCATCACCCTGCCATGCCGAATTAAGCAGTGCATTAATAT
		GTGGCAGCGGATAGGTCAGGCAATGTATGCTCCCCCAATCCAGGGAGTTATTCGG
		TGCGTCTCTAATATCACGGGACTTATCTTGACTCGCGACGGCGGCAGCACGAACA
		GCACAACAGAAACATTCCGGCCTGGGGGGGGAGACATGCGGGATAACTGGCGGA
		GTGAGCTGTACAAGTATAAAGTTGTTAAGATCGAATGA
HIV	53	MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLF
Envelope		CASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHED
protein		IISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRG
		KVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKC
		NNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQ
		LNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQI
		ASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEG
		SNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNN
		ESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFL
		GFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQL
		QARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWD
		REINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVG
		GLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLV
		NGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQE
		LKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
Influenza	54	MKAKLFVLFCAFNALKADTICVGYHANNSTDTVDTILKKNVTVTHSVNLLENTHNG
A		KLCSLNGKAPLQLENCNVAGWILGNPECDFLLTENSWSYIIETSNSRNGTCYPGEFTN
Hemagglutinin		YEELREQLSTVSSFERFEIFPKVTSWPSHETTKGTTVACSHSGANSFYRNLLWIVKKGD
		SYPKLSKSYTNNKGKEVLVIWGVHHPPTYSDQQTLYQNNHTYVSVGSSKYYQRFTPE
		IVARPKIREQAGRMNYYWLLLDQGDTITFEATGNLIAPWHAFALDKGSNSGIMMSDA
		HVRNCTTKCQTPHGALKSNLPFQNVHPITIGECPKYVKSTHLRMATGLRNIPSIQSRGL
		FGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADKKSTQIAIDGISNKVNSIIEKM
		NIQFTSVGKEFNNLEKRIENLNKKVDDGFLDVWTYNAELLILLENERTLDFHDFNVRN
		LYERVKSQLRNNAKEIGNGCFEFYHKCDNECMESVKNGTYNYPRYSEESKLNREKID
		GVKLESLGVHQILAIYSTVASSLILLVSLGAIVFWMCSNGSLQCRICI
Influenza	55	MKAKLFVLFCAFNALKADTICVGYHANNSTDTVDTILKKNVTVTHSVNLLENTHNG
B		KLCSLNGKAPLQLENCNVAGWILGNPECDFLLTENSWSYIIETSNSRNGTCYPGEFTN
IIemagglutinin		YEELREQLSTVSSFERFEIFPKVTSWPSHIETTKGTTVACSIISGANSFYRNLLWIVKKGD
		SYPKLSKSYTNNKGKEVLVIWGVHHPPTYSDQQTLYQNNHTYVSVGSSKYYQRFTPE
		IVARPKIREQAGRMNYYWLLLDQGDTITFEATGNLIAPWHAFALDKGSNSGIMMSDA
		HVRNCTTKCQTPHGALKSNLPFQNVHPITIGECPKYVKSTHLRMATGLRNIPSIQSRGL
		FGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADKKSTQIAIDGISNKVNSIIEKM
		NIQFTSVGKEFNNLEKRIENLNKKVDDGFLDVWTYNAELLILLENERTLDFHDFNVRN
		LYERVKSQLRNNAKEIGNGCFEFYHKCDNECMESVKNGTYNYPRYSEESKLNREKID
		GVKLESLGVHQILAIYSTVASSLILLVSLGAIVFWMCSNGSLQCRICI
SARS-	56	MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANL
CoV-2		KGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVKPVTSGCFPIMHDR
Spike		TKIRQLPNLLRGYENIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKSGFFATMAWA
		VPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSA
		NGVTTHYVSQIGSFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKV
		WCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLK
		LANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADL
		KSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIE
		LAVLISNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAG
		TFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMLAIFIVYMVSR
		DNVSCSICL
Hepatitis C	57	MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF
Virus		SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLL
E2		IVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF
		LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINI
		TRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA
		LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYA
		WNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI
		APGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER
		DISTEIYQAGSTPCNGVEGENCYFPLQSYGFQPINGVGYQPYRVVVLSFELLHAPATV
		CGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQT
		LEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS
		NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSL
		GAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSF
		CTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIE
		DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSAL
		LAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQFNSAIGKIQ
		DSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQI
		DRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM
		SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQR
		NFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDL
		GDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLI
		AIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
Respiratory	58	VDAETHVTGGNAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGW
Syncytial		LAGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPC
Virus F		GIVPAKSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFG
		CTWMNSTGFTKVCGAPPCVIGGVGNNTLLCPTDCFRKYPEATYSRCGSGPRITPRCM
		VDYPYRLWHYPCTINYTIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSELSP
		LLLSTTQWQVLPCSFTTLPALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEYVVLL
		FLLLADARVCSCLW
Ebola	59	MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE
virus		ISNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARREIPRFMNY
glycoprotein		TLNNAKKTNVTLSKKQKQQAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNG
		VSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVT
		TPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQL
		PLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ
		SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT
		KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFY
		DPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSL
		IAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0105] Fab-Fab geometry of an IgG antibody has a strong impact on the avidity conveyed by divalent binding, especially for antibodies with low monovalent affinity. For the divalent immunogens disclosed herein, antigenic spacing (the distance between the two antigens or epitopes thereof) is a key parameter considered for optimizing binding of a certain neutralizing antibody (e.g., a preferred neutralizing antibody and/or a broadly neutralizing antibody) to the antigens.

[0106] In some embodiments, the divalent immunogen provides a fixed and rigid spacing of the two antigens or epitopes thereof. In some embodiments, the divalent immunogen presents two antigens at a fixed antigenic distance. In some embodiments, the divalent immunogen provides an antigenic spacing of about 3 to about 20 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 7 to about 19 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 13 to about 16 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 18, or about 20 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 14 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 15 nm. In some embodiments, the divalent immunogen provides an antigenic spacing of about 16 nm.

[0107] In some embodiments, the divalent immunogen provides precise positioning of two antigens or epitopes thereof such that certain (e.g, preferred) BCRs or neutralizing antibodies are preferably bound to both antigens simultaneously. In some embodiments, the divalent immunogen provides antigenic spacing that allows a certain (e.g, preferred) BCR or neutralizing antibody to bind divalently with 1:1 stoichiometry to bind both antigens of the divalent immunogen) in a relaxed state.

[0108] The present disclosure provides divalent immunogens that provide precise positioning of two antigens or epitopes thereof such that certain (e.g, preferred) BCRs and/or neutralizing antibodies bind to both antigens simultaneously under the most relaxed conformations of the BCR or antibody. In some embodiments, this relaxed conformation occurs with a certain angle (Fab-Fab angle) between the two Fab domains of the BCR or antibody. In some embodiments, two Fab domains of the bound BCR and/or antibody assume an angle of about 20 degrees to about 150 degrees. In some embodiments, the two Fab domains of the bound BCR and/or antibody assume an angle of about 90 degrees to about 140 degrees. In some embodiments, the two Fab domains of the bound BCR and/or antibody assume an angle of about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150 degrees. In some embodiments, the two Fab domains of the bound BCR and/or antibody assume an angle of about 110, about 120, or about 130 degrees. In some embodiments, the two Fab domains of the bound BCR and/or antibody assume an angle of about 120 degrees.

[0109] The present disclosure also provides divalent immunogens that increase the binding avidity of certain (e.g., preferred) BCRs and/or antibodies. In some embodiments, the divalent immunogen increases the binding avidity of certain BCRs and/or antibodies due to the ability of the certain BCRs and/or antibodies to bind divalently (e.g., to bind to both antigens in the divalent immunogen). Divalent binding of the two antigens provides an avidity boost to certain BCRs and/or antibodies. This may be a particularly valuable mechanism for selectively binding certain BCRs and/or antibodies. For instance, this may be particularly valuable for BCRs and/or antibodies with low monovalent binding affinity.

[0110] In some embodiments, the binding avidity of certain BCRs and/or antibodies is at least 1.5, at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 400, at least 600, at least 800, or at least 1000-fold higher for the two antigens covalently linked to the scaffold compared to the same antigen unlinked to the scaffold. In some embodiments, the binding avidity of certain BCRs and/or antibodies is at least 1.5-fold higher for the two antigens covalently linked to the scaffold compared to the same antigen unlinked to the scaffold. In some embodiments, the binding avidity of certain BCRs and/or antibodies is at least 10-fold higher for the two antigens covalently linked to the scaffold compared to the same antigen unlinked to the scaffold. In some embodiments, the binding avidity of certain BCRs and/or antibodies is at least 100-fold higher for the two antigens covalently linked to the scaffold compared to the same antigen unlinked to the scaffold.

[0111] Monovalent and divalent binding of a BCR and/or antibody to the antigens of the divalent immunogen, avidity of a BCR and/or antibody for an antigen linked to a scaffold of a divalent immunogen, and/or avidity of a BCR and/or antibody not linked to a scaffold, may be measured by any method known in the art. For example, monovalent and divalent binding and/or avidity may be measured in a gel shift assay as shown in FIG. 5A and described in Example 2. Alternatively or additionally, avidity may be measured by surface plasmon resonance.

[0112] In some embodiments, the second engineered Fab mutation of the divalent immunogen fixes the distance between the antigens or epitopes thereof. In some embodiments, the second engineered Fab mutation modifies the interaction between the antigens or epitopes thereof and certain (e.g., preferred) BCRs and/or antibodies. In a non-limiting example, the second engineered Fab mutation may increase the avidity of certain (e.g., preferred) BCRs and/or antibodies for the antigens in the divalent immunogen. In a non-limiting example, the second engineered Fab mutation may decrease the avidity of other (e.g., non-preferred) BCRs and/or antibodies for the antigens in the divalent immunogen. In some embodiments, the second engineered Fab mutation enhances the binding avidity of certain BCRs and/or antibodies for the antigen or epitope thereof by at least 1.5-fold. In some embodiments, the second engineered Fab mutation enhances the binding avidity of certain BCRs and/or antibodies for the antigen or epitope thereof by at least 10-fold. In some embodiments, the second engineered Fab mutation enhances the binding avidity of certain BCRs and/or antibodies for the antigen or epitope thereof by at least 100-fold.

[0113] BCR crosslinking likely plays an important role in B-cell activation. The present disclosure provides multimers of the divalent immunogens describe herein. Multimerization at high or low valency may be effective for different divalent immunogen and/or different applications.

[0114] In some embodiments, the multimer is a dimer, a trimer, a tetramer, a 5-mer, a 6-mer, a 7-mer, an 8-mer, a 9-mer, or a 10-mer. In some embodiments, the multimer is a dimer, a trimer, or a tetramer.

[0115] The present disclosure also provides multimers of divalent immunogens conjugated onto a nanoparticle. A non-limiting example of a technology suitable for conjugating the divalent immunogens disclosed herein onto nanoparticles is the SpyCatcher/Spy Tag technology (Keeble et al., Proc Natl Acad Sci USA, 2019. 116(52): p. 26523-26533).

[0116] In some embodiments, multimers of divalent immunogens may be conjugated onto a nanoparticle by generating a SpyTagged divalent immunogen and contacting it with a SpyCatcher-VLP (Rahikainen et al. Angew Chem Int Ed Engl, 2021. 60(1): p. 321-330). In some embodiments, the SpyTagged divalent immunogen and SpyCatcher-VLP are mixed at an empirically determined molar ratio such that 1/3 of the SpyCatcher-VLP monomers are conjugated (VLP is made of 60 monomers of which 10 divalent constructs (20 SpyTags) can react).

[0117] In some embodiments, to generate dimers and trimers, two separate cultures are generated: one expressing the 15.6 kDa SpyCatcher at the divalent scaffold interface (heavy chain C-termini for Fab-based divalent immunogens) and one expressing a 50/50 combination of SpyTagged and untagged divalent complex resulting in a mixed species containing zero (25%), one (50%) or two (25%) SpyTags. Mixing of the two batches of divalent construct will result in dimers and trimers which can be separated by size exclusion chromatography (SEC) with Sephacryl-S300.

[0118] In some embodiments, dimers and/or trimers of the divalent immunogen maintain selective avidity and selective activation for preferred BCRs over low-affinity BCRs. In some embodiments, extensive multimerization (e.g., 10mer) of the divalent antigen results in loss of selective avidity/activation.

[0119] The present invention further contemplates a pharmaceutical composition comprising a divalent immunogen and one or more pharmaceutically acceptable diluent, carrier, or excipient.

[0120] The divalent immunogens can be combined with pharmaceutically-acceptable carriers, diluents, excipients and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In some embodiments, the pharmaceutical compositions are sterile.

[0121] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition is sterile and should be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0122] Sterile solutions can be prepared by incorporating the divalent immunogen (or encoding polynucleotide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0123] The pharmaceutical composition may additionally comprise an immunomodulatory component, such as an adjuvant. The adjuvant for administration in combination with a composition described herein may be administered before, concomitantly with, or after administration of said composition.

[0124] Non-limiting examples of adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, for example, WO 90/14837); saponin formulations, for example, such as QS21 and immunostimulatory complexes (ISCOMS) (see, for example, U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are polyribosinic:polyribocytidic acid (polyI:C), monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3 dMPL), CpG motif oligonucleotides, ADP-ribosylating bacterial toxins or their mutants, such as E. coli thermolabile enterotoxin LT, cholera toxin CT, etc.

[0125] The present disclosure also provides polynucleotides encoding the scaffold and two antigens or epitopes thereof described herein. In some embodiments, a polynucleotide encodes a scaffold or portion thereof, wherein the encoded scaffold provides precise positioning of two antigens or epitopes thereof such that only certain (e.g., preferred) BCRs and/or antibodies are preferably bound to both antigens simultaneously. In some embodiments, a polynucleotide encodes an antigen or epitope thereof described therein. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence and a second polynucleotide encodes a light chain sequence.

[0126] In some embodiments, the polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the heavy chain sequence of any one of SEQ ID NOs: 1, 17, 19, 25, 29, 33, 37, 41, 45, 49, or 51. In some embodiments, the polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the light chain sequence of any one of SEQ ID NOs: 3, 5, 7, 9, 11, 21, 23, 27, 31, 35, 39, 43, 47, and 62. In some embodiments, the polynucleotide comprises a sequence that encodes an antigen sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 13, 15, 54, 55, 56, 57, 58, 59, and 60.

[0127] In some embodiments, the polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the heavy chain sequence of a co-receptor antibody. In some embodiments, the polynucleotide comprises a sequence that encodes the heavy chain sequence of a co-receptor antibody. In some embodiments, the polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the light chain sequence of a co-receptor antibody. In some embodiments, the polynucleotide comprises a sequence that encodes the light chain sequence of a co-receptor antibody. In some embodiments, a first polynucleotide comprises a sequence that encodes the heavy chain sequence of a co-receptor antibody and a second polynucleotide comprises a sequence that encodes the light chain sequence of a co-receptor antibody.

[0128] In some embodiments, the co-receptor binding site antibody is 48d. In some embodiments, the polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the heavy chain sequence of the 48d antibody. In some embodiments, the polynucleotide comprises a sequence that encodes the heavy chain sequence of the 48d antibody. In some embodiments, the polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to the light chain sequence of the 48d antibody. In some embodiments, the polynucleotide comprises a sequence that encodes the light chain sequence of the 48d antibody. In some embodiments, a third and fourth polypeptide each comprise a sequence that encodes an antigen at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to an HIV gp120, Core, or epitope thereof.

[0129] In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and a second polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and a second polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 5. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and a second polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID No: 7. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and a second polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and a second polynucleotide comprises a sequence that encodes a light chain sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO11. In some embodiments, a third and fourth polypeptide each comprise a sequence that encodes an antigen sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, a third and fourth polypeptide each comprise a sequence that encodes an antigen sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 15.

[0130] In some embodiments, a first polynucleotide comprises a sequence that encodes the heavy chain sequence of the 48d antibody and a second polynucleotide comprises a sequence that encodes the light chain sequence of the 48d antibody. In some embodiments, a third and fourth polypeptide each comprise a sequence that encodes an HIV gp120 antigen, Core, or epitope thereof.

[0131] In some embodiments, the polynucleotide comprises a sequence that encodes a heavy chain sequence comprising any one of SEQ ID NOs: 1, 17, 19, 25, 29, 33, 37, 41, 45, 49, 51, 63, and 67. In some embodiments, the polynucleotide comprises a sequence that encodes a light chain sequence comprising any one of SEQ ID NOs: 3, 5, 7, 9, 11, 21, 23, 27, 31, 35, 39, 43, 47, 61, 65, and 69. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence comprising any one of SEQ ID NOs: 1, 17, 49, or 51 and a second polynucleotide comprises a sequence that encodes a light chain sequence comprising any one of SEQ ID NOs: 3, 5, 7, 9, 11, and 62. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 19 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 21. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 19 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 23. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 25 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 27. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 29 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 31. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 33 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 35. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 37 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 39. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 41 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 43. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 45 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 47. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 63 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 65. In some embodiments, a first polynucleotide comprises a sequence that encodes SEQ ID NO: 67 and a second polynucleotide comprises a sequence that encodes SEQ ID NO: 69. In some embodiments, a third and fourth polynucleotide each comprise a sequence that encodes SEQ ID NO: 13. In some embodiments, a third and fourth polynucleotide each comprise a sequence that encodes SEQ ID NO: 15.

[0132] In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence comprising SEQ ID NO: 1 and a second polynucleotide encodes a light chain sequence comprising SEQ ID NO: 5. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence comprising SEQ ID NO: 1 and a second polynucleotide encodes a light chain sequence comprising SEQ ID No: 7. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence comprising SEQ ID NO: 1 a second polynucleotide encodes a light chain sequence comprising 9. In some embodiments, a first polynucleotide comprises a sequence that encodes a heavy chain sequence comprising SEQ ID NO: 1 a second polynucleotide encodes a light chain sequence comprising SEQ ID NO: 11. In some embodiments, a third and fourth polynucleotide each comprise a sequence that encodes SEQ ID NO: 13. In some embodiments, a third and fourth polynucleotide each comprise a sequence that encodes SEQ ID NO: 15.

[0133] In some embodiments, the polynucleotides comprise a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 62, 64, 66, 68, and 70. In some embodiments, a first polynucleotide comprises a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to at least one of SEQ ID NOs: 2, 18, 20, 26, 30, 34, 38, 42, 46, 50, 52, 64, and 68, and a second polynucleotide comprises a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical to at least one of SEQ ID NOs: 4, 6, 8, 10, 12, 22, 24, 28, 32, 36, 40, 44, 48, 62, 66, and 70.

[0134] In some embodiments, the polynucleotides comprise at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 62, 64, 66, 68, and 70. In some embodiments, a first polynucleotide comprises any one of SEQ ID NOs: 2, 18, 20, 26, 30, 34, 38, 42, 46, 50, 52, 64, and 68, and a second polynucleotide comprises any one of SEQ ID NOs: 4, 6, 8, 10, 12, 22, 24, 28, 32, 36, 40, 44, 48, 62, 66, and 70.

[0135] In some embodiments, a first polynucleotide comprises SEQ ID NO: 2 and a second polynucleotide comprises SEQ ID NO: 4. In some embodiments, a first polynucleotide comprises SEQ ID NO: 2 and a second polynucleotide comprises SEQ ID NO: 6. In some embodiments, a first polynucleotide comprises SEQ ID NO: 2 and a second polynucleotide comprises SEQ ID NO: 8. In some embodiments, a first polynucleotide comprises SEQ ID NO: 2 and a second polynucleotide comprises SEQ ID NO: 10. In some embodiments, a first polynucleotide comprises SEQ ID NO: 2 and a second polynucleotide comprises SEQ ID NO: 12. In some embodiments, a first polynucleotide comprises SEQ ID NO: 2 and a second polynucleotide comprises SEQ ID NO: 62. In some embodiments, a third and fourth polynucleotide each comprise SEQ ID NO: 14. In some embodiments, a third and fourth polynucleotide each comprise SEQ ID NO: 16.

[0136] In some embodiments, a first polynucleotide comprises SEQ ID NO: 18 and a second polynucleotide comprises SEQ ID NO: 4. In some embodiments, a first polynucleotide comprises SEQ ID NO: 18 and a second polynucleotide comprises SEQ ID NO: 6. In some embodiments, a first polynucleotide comprises SEQ ID NO: 18 and a second polynucleotide comprises SEQ ID NO: 8. In some embodiments, a first polynucleotide comprises SEQ ID NO: 18 and a second polynucleotide comprises SEQ ID NO: 10. In some embodiments, a first polynucleotide comprises SEQ ID NO: 18 and a second polynucleotide comprises SEQ ID NO: 12.

[0137] In some embodiments, a first polynucleotide comprises SEQ ID NO: 20 and a second polynucleotide comprises SEQ ID NO: 22. In some embodiments, a first polynucleotide comprises SEQ ID NO: 20 and a second polynucleotide comprises SEQ ID NO: 24. In some embodiments, a first polynucleotide comprises SEQ ID NO: 26 and a second polynucleotide comprises SEQ ID NO: 28. In some embodiments, a first polynucleotide comprises SEQ ID NO: 30 and a second polynucleotide comprises SEQ ID NO: 32. In some embodiments, a first polynucleotide comprises SEQ ID NO: 34 and a second polynucleotide comprises SEQ ID NO: 36. In some embodiments, a first polynucleotide comprises SEQ ID NO: 38 and a second polynucleotide comprises SEQ ID NO: 40. In some embodiments, a first polynucleotide comprises SEQ ID NO: 42 and a second polynucleotide comprises SEQ ID NO: 44. In some embodiments, a first polynucleotide comprises SEQ ID NO: 46 and a second polynucleotide comprises SEQ ID NO: 48. In some embodiments, a first polynucleotide comprises SEQ ID NO: 64 and a second polynucleotide comprises SEQ ID NO: 66. In some embodiments, a first polynucleotide comprises SEQ ID NO: 68 and a second polynucleotide comprises SEQ ID NO: 70. In some embodiments, a third and fourth polynucleotide each comprise SEQ ID NO: 14. In some embodiments, a third and fourth polynucleotide each comprise SEQ ID NO: 16.

[0138] The present disclosure also provides cells comprising at least one polynucleotide described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell (e.g., a murine, bovine, simian, porcine, equine, ovine, or human cell). In some embodiments, the cell is a human cell.

[0139] Non-limiting examples of polynucleotide sequences that may encode divalent immunogen scaffolds are shown in Table 3 and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 18, 20, 22, 24, 26, 28, 30, 32, 24, 36, 38, 40, 42, 44, 46, 48, 50, 52, 62, 64, 66, 68, and 70.

TABLE 3
Non-limiting examples of sequences comprised by polynucleotides encoding
divalent immunogen scaffolds
	SEQ ID
ID	NO.	Sequence
48d	2	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
antibody		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
HC		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCA
		CACATGCCCACCGTGCCCAGCACCTAACGCTGCTGGTGGACCATCCGTCTTCA
		TCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTC
		ACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCT
		GGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGA
		GGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGG
		ACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCGG
		CGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCA
		CAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCA
		CTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGG
		ACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTG
		GACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGA
		ACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCA
		CAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTTGA
48d	4	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
antibody		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
LC		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCA
		ACAGAGGAGAATGCTGA
48d	6	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
antibody		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
LC K126C		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGTGCTCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCA
		ACAGAGGAGAATGCTGA
48d	8	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
antibody		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
LC Loop1		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAAACGATGA
		ACAGCTGCTGCCTTGCGCAGGGCCGAGCAACGCCTCTGTTGTGTGCCTGCTGA
		ATAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCT
		GCAGAGCGGAAACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTC
		CACATACAGCCTGAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAG
		CACAAGGTGTACGCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGA
		CAAAGAGCTTCAACAGAGGAGAATGCTGA
48d	10	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
antibody		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
LC Loop2		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAGCACCTCC
		TGAGGGGTGCGGGAGCCCTACTAGAACTGTTGTGTGCCTGCTGAATAACTTCT
		ACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCG
		GAAACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACA
		GCCTGAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGT
		GTACGCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGC
		TTCAACAGAGGAGAATGCTGA
48d	12	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
antibody		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
LC Loop3		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCAGAGCAGAGGAGG
		GGTGCGGGGGGAACACTGTGTACCTGGTGTGCCTGCTGAATAACTTCTACCCC
		AGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAAAC
		AGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCTGA
		GCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGC
		CTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCAAC
		AGAGGAGAATGCTGA
gp120.Core	18	ATGCCCATGGGTAGCCTGCAACCACTGGCCACCCTGTATCTGTTAGGGATGTT
48D + 48D.		GGTGGCCTCCGTGCTTGCAGATATCCGACAGGCCCATTGCAATGTATCTAAAG
Heavy_Fusion		CCACCTGGAATGAGACACTGGGTAAAGTCGTTAAGCAGTTGCGGAAACACTT
		TGGCAATAATACGATCATCCGGTTCGCTAACTCCTCCGGGGGAGACTTGGAG
		GTCACGACCCATTCTTTCAATTGCGGCGGAGAATTTTTTTATTGCAACACCTC
		AGGCCTGTTCAATTCTACCTGGATTTCAAATACTAGTGTGCAGGGATCTAACA
		GTACCGGTTCAAACGACAGCATAACATTGCCCTGTCGGATTAAACAGTGTATC
		AACATGTGGCAGCGCATAGGTCAGGCCATGTATGCCCCTCCTATTCAAGGAG
		TGATACGATGCGTGTCCAACATAACAGGACTTATTCTCACAAGAGATGGAGG
		ATCCACCAACTCTACGACTGAGACCTTTAGACCAGGAGGTGGAGACATGAGA
		GATAACTGGAGGAGTGAGCTTTACAAATACAAGGTCGTAAAAATCGAGGGGG
		GGGTGTGGAAAGATGCCGAGACTACGTTATTCTGCGCCTCAGACGCCAAGGC
		CTATGAAACAGAGAAACACAACGTGTGGGCAACCCATGCATGTGTTCCGACC
		GACCCCAATCCACAGGAGATCCACCTGGAAGGCGTCACAGAAGAATTCAACA
		TGTGGAAGAACAATATGGTGGAGCAGATGCACACCGATATTATTAGTCTGTG
		GGACCAATCACTGAAACCATGTGTCAAACTTACCGGCGGTAGCGCCATCACC
		CAGGCATGTCCTAAAGTAAGTTTCGAGCCAATCCCCATCCATTATTGCGCACC
		TGCCGGCTTCGCAATCCTGAAATGTAAGGATAAGAAGTTCAATGGCACAGGC
		CCTTGTCCGAGCGTGAGCACCGTCCAATGCACACACGGAATTAAACCTGTAG
		TCAGCACTCAGCTGCTGCTGAATGGCTCCCTGGCAGAAGAGGAAGTCATGAT
		CCGCTCAGAAAACATCACAAATAACGCTAAAAACATTCTGGTTCAATTTAAC
		ACGCCTGTCCAGATCAACTGTACCAGGCCGGGCAACGGAGGTGGTGGTGGCA
		GTGGGGGGGGTTCAGGTGGCGGCTCCGAAGTTCAACTTGTGCAGTCAGGAGC
		TGAGGTCAAGAAACCCGGTGCTACAGTGAAGATTAGCTGCAAAGCAAGCGGC
		TATACCTTTTCAGACTTTTATATGTACTGGGTTCGCCAAGCCCCCGGCAAAGG
		TCTGGAATGGATGGGCCTGATTGATCCAGAAGACGCCTGCACTATGTACGCA
		GAGAAATTCCGAGGTAGGGTTACTATCACTGCTGACACGTCCACCGATACAG
		GCTATCTAGAGCTTAGCTCTCTAAGAAGTGAAGACACGGCAGTGTATTATTGT
		GCTGCCGACCCATGGGAACTCAATGCCTTTAACGTTTGGGGACAAGGGACAC
		TTGTCAGCGTTTCAAGCGCCTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCA
		CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
		AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
		CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
		TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT
		CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGA
		GCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTAAC
		GCTGCTGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACT
		CATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAG
		GATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACA
		CAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGT
		CAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAA
		TGCAAGGTCAACAACAAAGACCTCGGCGCGCCCATCGAGAGAACCATCTCAA
		AACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGA
		AGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTC
		ATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAA
		ACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTAC
		AGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCC
		TGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTC
		CCGGACTCCGGGTTGA
RFD1_Heavy	20	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTACAGCTAATACAGAAGTGACCCCTGA
		GAGCGTGCGGGCCGCACTGCGCGAGCTGTTCGGAGATGAGGAACTGCTGGCC
		TGGGCCGCAGAGCTTCTGAGAGCCTGA
RFD1_Light	22	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
	24	CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAGTGTCCTACG
		AGAAGCTTGGACCCGGTCTGTGGATCTACCAGACTATTCAGGATGGCAAGCT
		CCTGTCAATTTCCACTAATGCAAAGCCCAGCACTCCACTGGAAGAGAAAGTG
		CGAGCTGTCGCCATCAGGGAGAAGCTTATCGCTGAGAACGCCGCCTGA
RFD1_Light.		ATGCCCATGGGCAGCTTACAGCCCCTTGCCACACTGTATCTCTTGGGGATGCT
gpFusion:		TGTTGCCTCCGTACTCGCCAGCGCAATCACTCAGGCTTGTCCAAAGGTTTCCT
		TTGAACCGATACCCATTCACTATTGTGCTCCCGCCGGCTTCGCCATCCTTAAG
		TGTAAGGACAAGAAATTCAATGGCACTGGCCCGTGTCCCTCCGTGTCCACCGT
		GCAGTGCACCCATGGCATAAAACCCGTGGTGAGCACTCAGCTGCTGCTGAAC
		GGTTCATTGGCCGAGGAAGAAGTTATGATCAGATCTGAGAACATCACGAATA
		ACGCTAAAAATATCCTGGTTCAGTTCAACACCCCAGTACAAATCAATTGTACT
		AGACCAGGCAACGGCGGCGACATCCGCCAGGCTCATTGTAACGTCTCCAAGG
		CAACTTGGAATGAAACACTCGGGAAAGTGGTGAAACAGCTGCGCAAGCATTT
		TGGGAATAACACCATCATACGATTCGCCAATAGCAGCGGGGGAGACCTGGAG
		GTTACCACTCACAGTTTCAACTGTGGAGGCGAATTTTTCTATTGTAACACATC
		AGGACTGTTCAATTCTACATGGATAAGTAACACATCAGTCCAGGGGTCAAAC
		AGTACCGGAAGTAATGACAGCATCACCCTGCCCTGTAGAATTAAGCAGTGCA
		TTAACATGTGGCAGCGGATCGGCCAGGCTATGTACGCCCCTCCCATACAGGG
		CGTTATCCGTTGCGTGAGCAATATTACAGGCTTGATTTTAACTAGAGACGGCG
		GTTCCACAAATTCTACAACCGAAACCTTCAGACCCGGCGGGGGCGACATGCG
		TGACAACTGGCGCTCAGAACTCTACAAATACAAAGTTGTGAAAATTGAGGGA
		GGCGTATGGAAGGACGCTGAAACCACCCTGTTCTGCGCAAGCGACGCCAAGG
		CATATGAAACTGAGAAACACAACGTATGGGCCACACACGCTTGTGTCCCCAC
		AGATCCCAACCCCCAAGAGATTCACCTCGAGGGTGTTACAGAGGAATTCAAC
		ATGTGGAAAAACAACATGGTCGAGCAGATGCACACAGACATCATATCCCTCT
		GGGATCAAAGTCTGAAGCCCTGCGTGAAACTGACTGGCGGCGGTAGTGGGGG
		TGGATCAGGCGGTGGTAGTGATATTCAAATGACGCAAAGTCCTAGCAGTGTC
		TCTGCCAGTGTGGGTGACCGAGTCACAATCACCTGTAGAGCTTCACAGGATAT
		TAGTACCTGGTTGGCTTGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTC
		CTGATTTACGCCGCCTCAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGG
		GAGCGGGTCTGGGACCGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAA
		GATTTTGCCACCTATTATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGG
		GGGAACTAAAGTTGAGATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCT
		TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
		CTGAATAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACG
		CCCTGCAGAGCGGAAACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGG
		ATTCCACATACAGCCTGAGCAGCACACTGACACTGTCCAAGGCCGACTACGA
		GAAGCACAAGGTGTACGCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCT
		GTGACAGTGTCCTACGAGAAGCTTGGACCCGGTCTGTGGATCTACCAGACTAT
		TCAGGATGGCAAGCTCCTGTCAATTTCCACTAATGCAAAGCCCAGCACTCCAC
		TGGAAGAGAAAGTGCGAGCTGTCGCCATCAGGGAGAAGCTTATCGCTGAGAA
		CGCCGCCTGA
RFD2_Heavy	26	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTTCTACTCCAGTCGAGGCAGTGCTTCC
		TCCCGAGGCACTTGCTCATTTGAGAGCTTTCCTGGCTCTGCTTCCGCTGGAGG
		CCGCTCTTGCCGTGGCTCACGCCGTGCGGCAGGCCCTGATCGCAGTCCAA
RFD2_Light	28	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAGCCAGAATCC
		TGAGGGTGAACGATGTAGTATTTACTTCTACCCAGACCGAAACATCTGGAGA
		GTGGACTGTTAACTCTACACCTCTGGATCAGCTCCTGCTTGATATCTACTTGG
		AGGCCCAGGCCGCC
RFD3_Heavy	30	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTGAGCCTAAAAACTCTGTGACACTGAC
		ATCCACGGCTGCCGACAGGGAGCTCGAGGAGGCATTGAGGAAACTGGCCCCC
		GAGGCCAAGTTTGTATTGGTCGGAACCCTGCCCGATGGCTCTACCCTCGTTAT
		TGTGTTTGTTTCC
RFD3_Light	32	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCA
		ATAAATATCCAGGCGTGGTGGTTACCGAAACAGTCGATGAGGCAACCGGCAC
		CACTACAATTACCCTCAACACTAAAAACTATAAGGCAGTTCGCCTGGCCATCA
		AAATTGCCGAAGCCCTGCATAAACTGCGTCCGGGCTCCACCACCGTTGCCGTT
		ATCAGT
RFD4_Heavy	34	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTCCCACAAACAAAGTGGTTACTGTGAC
		AATCGAGGTCAAGGAGGAGGAGCTTCCCGAGGACGTGTCTGTGAGGATTCGT
		ATGGAGATCGATATTAGTGAGAAGACAGGAAAGGTTGAAATGCGAATTGAA
		GTTAGCAAGAGC
RFD4_Light	36	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAGTTACCATCCA
		CAGGTTTAAGCCCAATAATGTGGTCACCATCGAGAAACTGCCCAGCGGGAAA
		ATCAAAGTGACCCTGACAGCTGAAAACGTTGAAGAAGCATTGGTTAACGCCT
		ACAGGCTGGCTCTTGAACTAGCAGAGGAG
RFD5_Heavy	38	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTGAACCCAAGAACTGGGTGAAGGCCA
		CTGTTAAAGTCGAGGACGGGGTGGTGGAAGCCGAGTTTGACTCCGGTGGGAG
		TAAAAACATCATTGAGGGACTGGCCAATTCTATGAAACTGCTCGCTGAGGTG
		GCTCTAGAGCTGCTCAGC
RFD5_Light	40	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGTCTTTTAA
		TAGGCTAAGTTCTGGAACATGGGCCACCATCGAGAAGAAGGAAGATCCCGGC
		GAGATGAACGTGACAGCCAACGTAGCAAACGTTATTAGCGCTAAGATCAAGG
		TCGAGTTCAAG
RFD6_Hcavy	42	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTGAGCCAAAAGAGGTGCTCAACCGCG
		TGACCGTGAAAGTGCCCGCCGAATTGACTGAGGCCGAAATTGCCATGCTCAA
		GACTCTCCTGAAAGCCCTCGGTGAGGCTATTGCTAAGGCCGCTAAA
RFD6_Light	44	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGTCCTTCA
		ACAAGGAGACCGGACTCGAGACAGATGTGGATCCCAAAGATGCAGACACTCC
		TGAGTACAAAAGGGTGACCGTCTATACTCCGCTGACCGCAGAGGATGTGGAA
		CTGTTTAAGAAGCTTGCTGAGAAGCTGGCCGAAGCCATCTATGAAGCCGCCA
		AAGAAAGG
RFD7_Heavy	46	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCCGAAGTTCAACTTGTGCAGTCAGGAGCTGAGGTCAAGAAACCCGGTGCT
		ACAGTGAAGATTAGCTGCAAAGCAAGCGGCTATACCTTTTCAGACTTTTATAT
		GTACTGGGTTCGCCAAGCCCCCGGCAAAGGTCTGGAATGGATGGGCCTGATT
		GATCCAGAAGACGCCTGCACTATGTACGCAGAGAAATTCCGAGGTAGGGTTA
		CTATCACTGCTGACACGTCCACCGATACAGGCTATCTAGAGCTTAGCTCTCTA
		AGAAGTGAAGACACGGCAGTGTATTATTGTGCTGCCGACCCATGGGAACTCA
		ATGCCTTTAACGTTTGGGGACAAGGGACACTTGTCAGCGTTTCAAGCGCCTCG
		ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
		GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
		ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
		CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
		TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
		GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAGAACGGCTTCAGTCTTTC
		TGGCTCCCCTGAAGAGGTGGTGGCACAGGCACTGAAGCATCATACCGGAGAT
		GAAAAGGTGGCCAAGATGGGCGTCGCACTAGTGAAGGAGCTGTTGGCT
RFD7_Light	48	ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACA
		TTCAGATATTCAAATGACGCAAAGTCCTAGCAGTGTCTCTGCCAGTGTGGGTG
		ACCGAGTCACAATCACCTGTAGAGCTTCACAGGATATTAGTACCTGGTTGGCT
		TGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTCCTGATTTACGCCGCCT
		CAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGGGAGCGGGTCTGGGAC
		CGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAAGATTTTGCCACCTATT
		ATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGGGGGAACTAAAGTTGAG
		ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACC
		CCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAA
		ACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCT
		GAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC
		GCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGTTTCA
		ACCGGTATCCCAGCGGCGTGATTGTATACAGGACGACCTCCAACGGCTCCAG
		CAGCTACGGCATTGATGCTAATCTGAAGACACTGGAAGCACTGCTGGAATTT
		ATCGAGGCAGTGGTTGAGATCTACAAAGAAAGG
48D H.C56D.	50	ATGCCCATGGGTAGCCTGCAACCACTGGCCACCCTGTATCTGTTAGGGATGTT
MouseIgG2a.		GGTGGCCTCCGTGCTTGCAGATATCCGACAGGCCCATTGCAATGTATCTAAAG
Kde1.Spy:		CCACCTGGAATGAGACACTGGGTAAAGTCGTTAAGCAGTTGCGGAAACACTT
		TGGCAATAATACGATCATCCGGTTCGCTAACTCCTCCGGGGGAGACTTGGAG
		GTCACGACCCATTCTTTCAATTGCGGCGGAGAATTTTTTTATTGCAACACCTC
		AGGCCTGTTCAATTCTACCTGGATTTCAAATACTAGTGTGCAGGGATCTAACA
		GTACCGGTTCAAACGACAGCATAACATTGCCCTGTCGGATTAAACAGTGTATC
		AACATGTGGCAGCGCATAGGTCAGGCCATGTATGCCCCTCCTATTCAAGGAG
		TGATACGATGCGTGTCCAACATAACAGGACTTATTCTCACAAGAGATGGAGG
		ATCCACCAACTCTACGACTGAGACCTTTAGACCAGGAGGTGGAGACATGAGA
		GATAACTGGAGGAGTGAGCTTTACAAATACAAGGTCGTAAAAATCGAGGGGG
		GGGTGTGGAAAGATGCCGAGACTACGTTATTCTGCGCCTCAGACGCCAAGGC
		CTATGAAACAGAGAAACACAACGTGTGGGCAACCCATGCATGTGTTCCGACC
		GACCCCAATCCACAGGAGATCCACCTGGAAGGCGTCACAGAAGAATTCAACA
		TGTGGAAGAACAATATGGTGGAGCAGATGCACACCGATATTATTAGTCTGTG
		GGACCAATCACTGAAACCATGTGTCAAACTTACCGGCGGTAGCGCCATCACC
		CAGGCATGTCCTAAAGTAAGTTTCGAGCCAATCCCCATCCATTATTGCGCACC
		TGCCGGCTTCGCAATCCTGAAATGTAAGGATAAGAAGTTCAATGGCACAGGC
		CCTTGTCCGAGCGTGAGCACCGTCCAATGCACACACGGAATTAAACCTGTAG
		TCAGCACTCAGCTGCTGCTGAATGGCTCCCTGGCAGAAGAGGAAGTCATGAT
		CCGCTCAGAAAACATCACAAATAACGCTAAAAACATTCTGGTTCAATTTAAC
		ACGCCTGTCCAGATCAACTGTACCAGGCCGGGCAACGGAGGTGGTGGTGGCA
		GTGGGGGGGGTTCAGGTGGCGGCTCCGAAGTTCAACTTGTGCAGTCAGGAGC
		TGAGGTCAAGAAACCCGGTGCTACAGTGAAGATTAGCTGCAAAGCAAGCGGC
		TATACCTTTTCAGACTTTTATATGTACTGGGTTCGCCAAGCCCCCGGCAAAGG
		TCTGGAATGGATGGGCCTGATTGATCCAGAAGACGCCTGCACTATGTACGCA
		GAGAAATTCCGAGGTAGGGTTACTATCACTGCTGACACGTCCACCGATACAG
		GCTATCTAGAGCTTAGCTCTCTAAGAAGTGAAGACACGGCAGTGTATTATTGT
		GCTGCCGACCCATGGGAACTCAATGCCTTTAACGTTTGGGGACAAGGGACAC
		TTGTCAGCGTTTCAAGCGCCTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCA
		CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
		AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
		CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
		TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT
		CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGA
		GCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTAAC
		GCTGCTGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACT
		CATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAG
		GATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACA
		CAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGT
		CAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAA
		TGCAAGGTCAACAACAAAGACCTCGGCGCGCCCATCGAGAGAACCATCTCAA
		AACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGA
		AGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTC
		ATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAA
		ACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTAC
		AGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCC
		TGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTC
		CCGGACTCCGGGTTGA
48D H.	52	ATGCCCATGGGTAGCCTGCAACCACTGGCCACCCTGTATCTGTTAGGGATGTT
ShortUpperHinge.		GGTGGCCTCCGTGCTTGCAGATATCCGACAGGCCCATTGCAATGTATCTAAAG
MouseIgG2a.		CCACCTGGAATGAGACACTGGGTAAAGTCGTTAAGCAGTTGCGGAAACACTT
Kde1.Spy:		TGGCAATAATACGATCATCCGGTTCGCTAACTCCTCCGGGGGAGACTTGGAG
		GTCACGACCCATTCTTTCAATTGCGGCGGAGAATTTTTTTATTGCAACACCTC
		AGGCCTGTTCAATTCTACCTGGATTTCAAATACTAGTGTGCAGGGATCTAACA
		GTACCGGTTCAAACGACAGCATAACATTGCCCTGTCGGATTAAACAGTGTATC
		AACATGTGGCAGCGCATAGGTCAGGCCATGTATGCCCCTCCTATTCAAGGAG
		TGATACGATGCGTGTCCAACATAACAGGACTTATTCTCACAAGAGATGGAGG
		ATCCACCAACTCTACGACTGAGACCTTTAGACCAGGAGGTGGAGACATGAGA
		GATAACTGGAGGAGTGAGCTTTACAAATACAAGGTCGTAAAAATCGAGGGGG
		GGGTGTGGAAAGATGCCGAGACTACGTTATTCTGCGCCTCAGACGCCAAGGC
		CTATGAAACAGAGAAACACAACGTGTGGGCAACCCATGCATGTGTTCCGACC
		GACCCCAATCCACAGGAGATCCACCTGGAAGGCGTCACAGAAGAATTCAACA
		TGTGGAAGAACAATATGGTGGAGCAGATGCACACCGATATTATTAGTCTGTG
		GGACCAATCACTGAAACCATGTGTCAAACTTACCGGCGGTAGCGCCATCACC
		CAGGCATGTCCTAAAGTAAGTTTCGAGCCAATCCCCATCCATTATTGCGCACC
		TGCCGGCTTCGCAATCCTGAAATGTAAGGATAAGAAGTTCAATGGCACAGGC
		CCTTGTCCGAGCGTGAGCACCGTCCAATGCACACACGGAATTAAACCTGTAG
		TCAGCACTCAGCTGCTGCTGAATGGCTCCCTGGCAGAAGAGGAAGTCATGAT
		CCGCTCAGAAAACATCACAAATAACGCTAAAAACATTCTGGTTCAATTTAAC
		ACGCCTGTCCAGATCAACTGTACCAGGCCGGGCAACGGAGGTGGTGGTGGCA
		GTGGGGGGGGTTCAGGTGGCGGCTCCGAAGTTCAACTTGTGCAGTCAGGAGC
		TGAGGTCAAGAAACCCGGTGCTACAGTGAAGATTAGCTGCAAAGCAAGCGGC
		TATACCTTTTCAGACTTTTATATGTACTGGGTTCGCCAAGCCCCCGGCAAAGG
		TCTGGAATGGATGGGCCTGATTGATCCAGAAGACGCCTGCACTATGTACGCA
		GAGAAATTCCGAGGTAGGGTTACTATCACTGCTGACACGTCCACCGATACAG
		GCTATCTAGAGCTTAGCTCTCTAAGAAGTGAAGACACGGCAGTGTATTATTGT
		GCTGCCGACCCATGGGAACTCAATGCCTTTAACGTTTGGGGACAAGGGACAC
		TTGTCAGCGTTTCAAGCGCCTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCA
		CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
		AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
		CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
		TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT
		CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGA
		GCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTAAC
		GCTGCTGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACT
		CATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAG
		GATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACA
		CAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGT
		CAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAA
		TGCAAGGTCAACAACAAAGACCTCGGCGCGCCCATCGAGAGAACCATCTCAA
		AACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGA
		AGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTC
		ATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAA
		ACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTAC
		AGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCC
		TGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTC
		CCGGACTCCGGGTTGA
gp120.Core	62	ATGCCCATGGGCAGCTTACAGCCCCTTGCCACACTGTATCTCTTGGGGATGCT
48D + 48D.		TGTTGCCTCCGTACTCGCCAGCGCAATCACTCAGGCTTGTCCAAAGGTTTCCT
Light_Fusion		TTGAACCGATACCCATTCACTATTGTGCTCCCGCCGGCTTCGCCATCCTTAAG
		TGTAAGGACAAGAAATTCAATGGCACTGGCCCGTGTCCCTCCGTGTCCACCGT
		GCAGTGCACCCATGGCATAAAACCCGTGGTGAGCACTCAGCTGCTGCTGAAC
		GGTTCATTGGCCGAGGAAGAAGTTATGATCAGATCTGAGAACATCACGAATA
		ACGCTAAAAATATCCTGGTTCAGTTCAACACCCCAGTACAAATCAATTGTACT
		AGACCAGGCAACGGCGGCGACATCCGCCAGGCTCATTGTAACGTCTCCAAGG
		CAACTTGGAATGAAACACTCGGGAAAGTGGTGAAACAGCTGCGCAAGCATTT
		TGGGAATAACACCATCATACGATTCGCCAATAGCAGCGGGGGAGACCTGGAG
		GTTACCACTCACAGTTTCAACTGTGGAGGCGAATTTTTCTATTGTAACACATC
		AGGACTGTTCAATTCTACATGGATAAGTAACACATCAGTCCAGGGGTCAAAC
		AGTACCGGAAGTAATGACAGCATCACCCTGCCCTGTAGAATTAAGCAGTGCA
		TTAACATGTGGCAGCGGATCGGCCAGGCTATGTACGCCCCTCCCATACAGGG
		CGTTATCCGTTGCGTGAGCAATATTACAGGCTTGATTTTAACTAGAGACGGCG
		GTTCCACAAATTCTACAACCGAAACCTTCAGACCCGGCGGGGGCGACATGCG
		TGACAACTGGCGCTCAGAACTCTACAAATACAAAGTTGTGAAAATTGAGGGA
		GGCGTATGGAAGGACGCTGAAACCACCCTGTTCTGCGCAAGCGACGCCAAGG
		CATATGAAACTGAGAAACACAACGTATGGGCCACACACGCTTGTGTCCCCAC
		AGATCCCAACCCCCAAGAGATTCACCTCGAGGGTGTTACAGAGGAATTCAAC
		ATGTGGAAAAACAACATGGTCGAGCAGATGCACACAGACATCATATCCCTCT
		GGGATCAAAGTCTGAAGCCCTGCGTGAAACTGACTGGCGGCGGTAGTGGGGG
		TGGATCAGGCGGTGGTAGTGATATTCAAATGACGCAAAGTCCTAGCAGTGTC
		TCTGCCAGTGTGGGTGACCGAGTCACAATCACCTGTAGAGCTTCACAGGATAT
		TAGTACCTGGTTGGCTTGGTATCAACAAAAGCCTGGAAAGGCCCCAAAACTC
		CTGATTTACGCCGCCTCAACATTGCAATCAGGAGTGCCTTCTAGATTTAGTGG
		GAGCGGGTCTGGGACCGACTTCTCACTTACAATTAACAGCCTGCAGCCGGAA
		GATTTTGCCACCTATTATTGCCAACAAGCTAACTCTTTCTTCACGTTCGGGGG
		GGGAACTAAAGTTGAGATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCT
		TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
		CTGAATAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACG
		CCCTGCAGAGCGGAAACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGG
		ATTCCACATACAGCCTGAGCAGCACACTGACACTGTCCAAGGCCGACTACGA
		GAAGCACAAGGTGTACGCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCT
		GTGACAAAGAGCTTCAACAGAGGAGAATGCTGA
RFD2_v2_	64	ATGGGCTGGTCCTGCATTATCCTGTTCCTGGTAGCGACAGCTACAGGTGTCCA
Heavy		CTCTGAAGTGCAGCTTGTCCAGAGTGGCGCCGAAGTTAAGAAACCCGGAGCC
		ACTGTGAAGATATCTTGTAAAGCTAGCGGATACACTTTCAGTGATTTTTATAT
		GTATTGGGTGAGGCAAGCCCCTGGTAAGGGCCTGGAGTGGATGGGCCTCATC
		GACCCAGAAGACGCCTGCACCATGTACGCTGAGAAGTTCCGAGGACGCGTCA
		CAATTACAGCAGATACATCTACGGATACGGGATACCTTGAACTTAGCTCCCTG
		CGGTCTGAGGATACCGCCGTGTATTATTGTGCAGCTGACCCCTGGGAACTCAA
		CGCTTTTAACGTATGGGGCCAAGGAACACTCGTATCTGTTAGCGGTGCCGCGG
		TGACTGGGCCACGAGTTTTTCCCCTGAGCCCAGCTGGCTGTTCTACCACATCA
		CCAACTACACTCCTGGGTTGCCTGGTCGACGACTACTACCCCCTGCCCGTTAC
		TGTAACCTGGAATGGCGGCGCTCTGACATCCGGCGTAACAACTTTCCCCGCCG
		TCCTGCGGCCTGACGGCTTGTACAGCCAGGTGTCCGTGCTGAGCGTCCCCTCT
		AGTAGCTGTGGCACAGAGACCTATACCTGCAACGTGACTCATCCAGCCTCCA
		ACACAACCGTTTCACAGCTGGTGCCCTGCCCAAGACAACTGGAATTGCCGGC
		CGAGGCCCTGGCGCACCTCAGGGCATTCCTGGCCTTGCTTCCGCCAGCCGCTG
		CCCTTGCAGTCGCACATGCGGTACTCCAGGCACTCGCAGCCGCTCTTTAA
RFD2_v2_	66	ATGGGATGGTCATGTATTATCCTTTTCCTGGTAGCCACTGCCACAGGAGTCCA
Light		CAGCGACATCCAGATGACCCAGTCCCCATCTTCCGTTTCTGCTAGTGTCGGAG
		ATAGAGTGACCATCACCTGCAGGGCCTCACAGGATATAAGTACGTGGCTGGC
		CTGGTACCAGCAGAAACCCGGGAAGGCCCCTAAATTGCTGATTTACGCCGCC
		TCCACACTCCAATCTGGGGTGCCTAGCCGGTTTTCCGGCAGTGGCAGCGGCAC
		GGATTTCAGTCTGACCATCAACTCACTACAGCCTGAGGACTTTGCCACATACT
		ACTGCCAGCAGGCCAACAGCTTCTTCACCTTTGGCGGAGGCACAAAGGTTGA
		GATCAAGCGCACCGTCGTCGCCCCTAGTGTGAGCATCTTCCCCCCTAGCCCTG
		AGGAGCTGGCATCCGGCCGCGCAAGGGTCGTGTGTGTAGCTTCCTCCTTTTAC
		CCCGAGTCTCTGGAAGTTACCTGGCTGGTGGACGGAGAGCCAGTGAGCGGAA
		GGTCTTTCACATCTACTTCAGCCAAGAACCCAGCAGACAATACCTATTCACTG
		GTCTCAACCTTGGATCTTTCTACAGAAGAGTATCTGGCCCATAGGCGCTTTGA
		ATGTAGAGTGACACATTCAGGCCTGAGCAGTCCTGTCACAGCCAGCATACTG
		AGGGTCGGGGACACCCTGTTCACTTCTGTTTCTTCTTCTGATGATGGAGTGTTT
		ACAGTCACCGGGTCTCCTCTGATGCAGCTGGCTCTGGATTTGTACTTGGAGGC
		TCAGGCCGCCTAG
RFD3_v2_	68	ATGGGGTGGTCCTGCATTATTCTGTTTTTGGTGGCTACCGCCACTGGAGTGCA
Heavy		CAGCGAAGTACAGCTTGTGCAAAGTGGTGCTGAGGTGAAAAAGCCAGGCGCA
		ACCGTGAAAATCAGCTGTAAGGCGAGCGGCTATACCTTCAGCGATTTCTACAT
		GTACTGGGTGCGGCAGGCACCTGGTAAAGGACTGGAGTGGATGGGTCTGATC
		GATCCAGAGGACGCCTGTACTATGTACGCGGAAAAATTTAGGGGTAGAGTGA
		CCATTACCGCAGATACCTCAACTGATACTGGCTATCTGGAACTTTCAAGTTTG
		CGCAGCGAAGATACCGCTGTCTACTATTGTGCAGCTGACCCCTGGGAACTTAA
		CGCATTCAACGTGTGGGGGCAGGGAACATTGGTCTCAGTCTCCGGCGCTCCC
		GTGACCGGGCCCGAGGTGTTTCCACTGCCACCTATGAGCGTAGGTAGCGGTA
		AGCCAAGCGCAACCCTGGGGTGCCTCGTTAAGAACTTTTATCCACTGCCTGTC
		GAAGTTACCTGGAATGGCGGCGCTGTCACAGAAGGTGTGTATGTGTTCCCTCC
		GGTATTGCGGCCAGATGGCAGATGGAGTCTCGTCAGCGTGCTGAGGGTTCCG
		GAGTCCACTCTTGGAACGGAGACATACGTGTGTAACGTGCGACATCCTCCAG
		CGAATGTTACAGTGAGCAAAGAAGTGAAGGCTCTACCAGGTGTAACTCTCAC
		ACAGACCCCTGAGGATGCTGCACTGGAAGCTAAGTTGAGGAAGGCCTGCCCT
		GAAGCAGAAGCAATTATAGTGGGGACATTGCCTAACGGCGCGACCCTGGTCT
		TGGTGTTTGTGCCCTGA
RFD3_v2_	70	ATGGGATGGTCTTGCATCATTCTGTTTCTGGTGGCTACCGCTACTGGGGTACA
Light		CAGCGATATACAGATGACCCAATCTCCCAGTTCCGTGTCCGCCTCCGTCGGAG
		ATCGTGTGACTATTACATGCCGCGCTAGCCAAGACATCAGTACATGGCTAGC
		ATGGTACCAGCAGAAGCCCGGGAAAGCACCCAAGCTTCTGATCTATGCAGCA
		TCCACCCTGCAGTCAGGAGTGCCCAGCCGCTTTTCCGGAAGCGGGAGTGGAA
		CCGATTTTTCTCTTACGATCAACTCTCTACAGCCCGAAGACTTCGCCACCTATT
		ATTGTCAGCAGGCGAACAGTTTTTTCACATTTGGTGGCGGCACTAAGGTGGAA
		ATCAAACGTACAGTGGCTGCTCCGTCCGTGTTCATCTTCCCCCCATCAGATGA
		GCAGCTGAAATCTGGCACCGCTAGCGTGGTCTGTCTTCTGAACAATTTCTACC
		CTCGCGAGGCTAAAGTCCAGTGGAAAGTTGACAACGCTCTGCAAAGCGGTAA
		TTCCCAGGAATCTGTTACAGAGCAGGACTCCAAGGATAGCACATATTCCCTG
		AGTAGTACCCTGACACTCAGCAAGGCCGACTACGAGAAGCATAAGGTTTACG
		CGTGTGAAGTAACCCACCAGGGTTTGAGTAGCCCCGTTACCAAAAGTTTCAAT
		AAGTATCCGGGCGTGGTGGTTACTGAAACCGTGGACGAGGCAACAGGTACCA
		CTACTATTACTCTTAACACGAAGAACTACAAGGCAGTCCGTCTCGCCATAAAG
		ATAGCCGAGGCTCTGCATAAGCTCAGGCCAGGTTCTACTACTGTTGCTGTCAT
		TTCTTGA

[0140] The present disclosure also provides methods of designing divalent immunogens described herein. In some embodiments, the method comprises first identifying a target bnAb. In other embodiments, the method comprises first positioning conserved epitopes of the antigen in such a way that the epitopes would engage both Fabs of a theoretical or hypothesized bnAb simultaneously and with 1:1 stoichiometry.

[0141] In some embodiments, the scaffold comprises a partially de novo engineered protein scaffold. Scaffolds comprising a partially de novo engineered protein scaffold may be based on a monomeric, dimeric, or multimeric protein. Design of such a scaffold based on a dimeric or multimeric protein comprises several steps.

[0142] First, a target bnAb is identified. Second, two antigens or epitopes are modeled such that the target broadly neutralizing antibody (bnAb) will engage them with both Fabs simultaneously. Third, a dimeric protein or multimeric is modeled (structure obtained from the Protein Data Bank or structure prediction software) wherein the dimeric or multimeric protein is centrally positioned with respect to the two antigens without spatial overlap. Portions of the dimeric or multimeric protein that may interfere with grafting of the modeled antigens onto it are removed, and/or the protein is extended as required to achieve optimal spacing of the antigens. Fourth, for a rotationally symmetrical model including the modeled antigens and the dimeric or multimeric protein, protein design tools are used to connect a monomer (of the dimeric or multimeric protein) to a single antigen/epitope within the model. For a non-symmetrical model, protein design tools are used to connect each monomer of the dimeric protein to each of the antigens/epitopes. In either case, it is critical to avoid spatial overlap between the partially de novo scaffold and modeled bnAb Fabs. Non-limiting examples of divalent immunogens designed by this method are shown in FIG. 16 and FIG. 17 and in SEQ ID NOs: 71 and 72.

[0143] Design of such a scaffold based on a monomeric protein comprises several steps. First, a target bnAb is identified. Second, two antigens or epitopes are modeled such that the target broadly neutralizing antibody (bnAb) will engage them with both Fabs simultaneously. Third, a monomeric scaffold is modeled near the two antigens or epitopes. Fourth, protein design tools are used to create peptide linkages between the antigens or epitopes and the monomeric scaffold, avoiding overlap between the partially de novo scaffold and modeled bnAb Fabs.

[0144] In other embodiments, the scaffold comprises a completely de novo engineered protein scaffold. Scaffolds comprising a completely de novo engineered protein scaffold may be based on a monomeric, dimeric, or multimeric protein. First, a target bnAb is identified. Second, two antigens or epitopes are modeled such that the target bnAb will engage them with both Fabs simultaneously. Third, protein design tools are used to generate a de novo scaffold linking the two antigens, avoiding spatial overlap with the modeled bnAb Fabs. This de novo scaffold may include a dimerization domain.

[0145] In designing a scaffold comprising either a partially or fully de novo engineered protein scaffold, there are several considerations. In some embodiments, an essential feature is limitation of rotational or translational freedom of the antigen/epitope in the model, such that the broadly neutralizing antibody has a rare ability of simultaneous Fab engagement, compared with other antibodies that bind to other epitopes or with different angles of approach.

[0146] The antigen or epitope thereof may be incorporated through any method known in the art. The epitope may be grafted onto the scaffold (e.g., as in FIG. 12A) or alternatively, the entire antigen may be included (e.g., as in FIG. 12B) or a reduced (Core) antigen that includes the epitope may be included. In one non-limiting example, the antigen, the epitope, or the Core is linked to the scaffold-two separate proteins-using disulfide bonding or another covalent linkage. In a second non-limiting example, the antigen, the epitope, or the Core is fused to the scaffold such that it is one contiguous protein that folds into essentially the “scaffold” domain and the “antigen/epitope” domain, wherein these domains come together in the desired positioning upon completion of folding. In a third non-limiting example, the antigen, the epitope, or the Core is fully integrated into the scaffold such that there is less of a distinction between the “scaffold” domain and the “antigen/epitope” domain. Optionally, it may be closer to an integrated scaffold+epitope protein.

[0147] Divalent immunogen designs may be optimized by any method known in the art. In a non-limiting example, scaffold designs are generated using AI-based protein design tools. In a second, non-mutually-exclusive example, linkages of the scaffold to the antigens, Cores, or epitopes of interest are generated using AI-based protein design tools. Using this approach can achieve multiple important design goals, including:

• • [0148] Optimized positioning and idealized geometry of the two copies of the antigen/epitope for high avidity engagement with the target bnAb;
  • [0149] Rigid structure which increases the specificity of the divalent binding to the target bnAb—the “selective avidity effect”;
  • [0150] Possible incorporation of linear epitopes or other desirable features into the design;
  • [0151] Addition of any tags or domains required for multimerization of the divalent antigen;
  • [0152] Minimized off-target immunogenicity;
  • [0153] Optimized expression of the protein; and/or
  • [0154] Optimized purification method.

[0155] The present disclosure also provides methods that comprise first positioning conserved epitopes of the antigen in such a way that the epitopes would engage both Fabs of a theoretical or hypothesized bnAb simultaneously and with 1:1 stoichiometry. In some embodiments, the method comprises first identifying a “theoretical” or “hypothesized” template bnAb that would engage conserved epitopes at a particular angle of approach. In some embodiments, a divalent immunogen modeled thereon could elicit previously undiscovered and uncharacterized bnAbs with similar epitopes and angles of approach as the template bnAb.

[0156] First, the conserved epitopes of the antigen are positioned in such a way that they would engage both Fabs of such a theoretical or hypothesized bnAb simultaneously and with 1:1 stoichiometry. In some embodiments, the positioning of the epitopes provides rotational and positional flexibility. In some embodiments, the epitopes are about 3 to about 20 nm apart (i.e., have an epitopic distance of about 3 to about 20 nm). In some embodiments, the epitopes are about 7 to about 19 nm apart (i.e., have an epitopic distance of about 7 to about 19 nm). In some embodiments, the epitopes are about 13 to about 16 nm apart (i.e., have an epitopic distance of about 13 to about 16 nm). In some embodiments, the epitopes are positioned such that the two Fab domains of a bound BCR assume an angle of about 20 to about 150 degrees. In some embodiments, the epitopes are positioned such that the two Fab domains of a bound BCR assume an angle of about 90 to about 140 degrees. In some embodiments, the epitopes are positioned such that the two Fab domains of a bound BCR assume an angle of about 120 degrees.

[0157] This would be an alternative way of completing “Step 2” in the methods to design a divalent immunogen listed previously. Subsequent steps of completing the design would remain the same.

[0158] The present disclosure also provides methods of identifying antibodies with preferred characteristics for divalent immunogens, comprising: (a) providing a divalent immunogen comprising a known broadly neutralizing antibody and an antigen or epitope thereof; (b) administering the divalent immunogen to a subject, optionally a mouse; (c) isolating B cells from the subject; (d) screening the isolated B cells for ability to bind to the divalent immunogen in high avidity, bidentate fashion; and (e) sequencing the complementarity determining regions of B cells that bind as in (d).

[0159] In some embodiments, the method comprises designing and providing an immune complex comprising a known broadly neutralizing antibody as a scaffold, and two antigens or epitopes thereof. In some embodiments, the method comprises administering the complex to an animal. In some embodiments, the complex is non-covalently or covalently cross-linked via chemical crosslinkers, engineered disulfide bonds, or expression of antibody-antigen chimeras. In some embodiments, the method activates/selects B cells whose receptors bind to the complex with high avidity. In some embodiments, the B cells undergo/continue affinity maturation.

[0160] In some embodiments, the B cells are isolated from the animal and screened for their ability to bind to the broadly neutralizing antibody-antigen complex in high avidity, bidentate fashion. In some embodiments, fluorescence-activated cell-sorting is used to isolate B-cells that preferentially bind to fluorophore-labeled intact broadly neutralizing antibody-antigen complex over a differentially labeled, fragmented bnAb-antigen complex. In some embodiments, the complementarity determining regions of these B cells will be sequenced, cloned and expressed to confirm the interaction using biochemical assays.

[0161] In some embodiments, once isolated from B-cells, the ideal antibody can be formulated into a divalent immunogen vaccine by complexation with the antigen. In some embodiments, the complexation may be non-covalent, or covalent in the form of chemical crosslinking, disulfide bond engineering or chimeric protein expression. In some embodiments, further mutations/chemical alterations to the antibody can be performed to lock the Fabs in a conformation that allows minimal entropic penalty in forming the ring-like structure with the BCR. In some embodiments, these modifications will also promote immune focusing by limiting the number of epitopes compatible with bidentate binding. In some embodiments, all or parts of the antibody may be “humanized” in order to minimize immunogenicity of the antibody alone. In some embodiments, the immune complex vaccine may be beneficially used to activate naïve B cells or may be used for further differentiation toward the desired specificity/breadth of neutralization of antibodies from B-cells already undergoing affinity maturation.

[0162] The present disclosure provides vaccines comprising at least one of the divalent immunogens described herein. In some embodiments, the vaccine comprises or consists of at least one multimer of a divalent immunogen described herein. In some embodiments, the vaccine comprises or consists of a pharmaceutical composition described herein. In some embodiments, the vaccine is administered to a subject. A subject may be any mammal, including non-human primate and human subjects. Non-limiting examples of a subject are a mouse and a human.

[0163] The present disclosure also provides methods of generating an immune response in a subject. In some embodiments, these methods comprise administering at least one divalent immunogen, at least one multimer, or at least one pharmaceutical composition disclosed herein, to a subject. The subject may be any mammal, including non-human primate and human subjects. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

[0164] The divalent immunogens and/or pharmaceutical compositions disclosed herein can be administered to a subject by any mode of delivery, including, for example, by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (e.g. see WO99/27961) or transcutaneous (e.g. see WO02/074244 and WO02/064162), intranasal (e.g. see WO03/028760), ocular, aural, pulmonary or other mucosal administration and/or inhalation of powder compositions. Multiple doses can be administered by the same or different routes.

[0165] The divalent immunogens, multimers, and/or pharmaceutical compositions disclosed herein can be administered prior to, concurrent with, or subsequent to delivery of other vaccines. Dosage with the divalent immunogens and pharmaceutical compositions may be a single dose schedule or a multiple dose schedule. A multiple dose schedule is one in which a course of vaccination may comprise 1-5 separate doses, in which a first “prime” dose is followed by other doses given at subsequent time intervals, chosen to maintain and/or reinforce the immune response. Non-limiting examples of time intervals between doses include 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, or 6 months. The dosage regimen will also, at least in part, be determined by the potency of the modality, the vaccine delivery employed, the need of the subject and be dependent on the judgment of the practitioner.

[0166] In some embodiments, the divalent immunogens and pharmaceutical compositions may be administered as “boost” vaccine dose after administration of a “prime” vaccine dose. The “prime” dose may be a different vaccine or may be a dose of a divalent immunogen or pharmaceutical composition of the present application. In other embodiments, the divalent immunogens and pharmaceutical compositions may be administered as a “prime” vaccine. One or more additional doses of a divalent immunogen or pharmaceutical composition of the present application, or of a different vaccine, may follow administration of the divalent immunogen and pharmaceutical composition. Also, the site of divalent immunogens or pharmaceutical composition administration may be the same or different as other vaccine compositions that are being administered.

[0167] In some embodiments, the divalent immunogen, multimer, pharmaceutical composition, or vaccine disclosed herein is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a specific antigen). “An effective amount” is a dose of the divalent immunogen, multimer, or pharmaceutical composition sufficient to produce an antigen-specific immune response.

[0168] In some embodiments, the immune response to a divalent immunogen, multimer, or pharmaceutical composition disclosed herein is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the administered divalent immunogen, multimer, or pharmaceutical composition.

[0169] In some embodiments, the immune response results in an increased level (titer) of a certain antibody that binds an antigen or epitope thereof present in the divalent immunogen, multimer, or pharmaceutical composition that was administered. In some embodiments, the certain antibody is a neutralizing antibody. In some embodiments, the neutralizing antibody is a bnAb (e.g., a preferred bnAb).

[0170] In some embodiments, the immune response results in an increased number of B cells expressing a BCR that binds an antigen or epitope thereof present in the divalent immunogen, multimer, or pharmaceutical composition that was administered. In some embodiments, the BCR binds the divalent immunogen divalently with increased avidity. In some embodiments, the BCR binds both antigens of the divalent immunogen simultaneously with 1:1 stoichiometry. In some embodiments, the BCR binds both antigens of the divalent immunogen simultaneously, wherein the two Fab domains of the bound BCR assume an angle (Fab-Fab angle) disclosed herein. In some embodiments, the immune response results in an increased number of B cells expressing a certain (e.g., a preferred) BCR. In some embodiments, the immune response results in an increased number of B cells expressing a VRC01-class BCR.

[0171] An immune response generated by administering at least one divalent immunogen, multimer, and/or pharmaceutical composition disclosed herein may be measured by any suitable method known in the art. In some embodiments, the immune response is assessed by determining the titer of a certain bnAb (e.g., a preferred bnAb) in the subject. In other embodiments, the immune response is assessed by measuring the quantity and/or characteristics of B cells expressing a certain BCR (e.g., a preferred BCR) in the subject. In other embodiments, the ability of serum or antibody from an immunized subject is tested for its ability to neutralize viral uptake.

[0172] Measurement of the antibody titer, B cells, and/or neutralizing capacity may be quantified with respect to a control. In some embodiments, the antibody titer, B cell quantity/characteristics, and/or neutralizing capacity may be measured and compared to level(s) in the same subject prior to administering the divalent immunogen, multimer, or pharmaceutical composition. In some embodiments, the antibody titer, B cell quantity/characteristics, and/or neutralizing capacity may be measured and compared to level(s) in subjects administered a control substance (e.g., a placebo).

[0173] An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a certain antigen or epitope of an antigen. In some embodiments, antibody titer is expressed as the inverse of the greatest dilution that provides a positive result. A non-limiting example of an assay for determining antibody titers is enzyme-linked immunosorbent assay (ELISA).

[0174] The quantity and/or characteristics of B cells expressing a certain (e.g., preferred) BCR may be assessed by metrics including, but not limited to, the number of B cells expressing a certain BCR, the number of B cells that recognize a certain antigen and/or divalent immunogen (e.g., through use of a specific probe), and surface markers of the B cells that indicate phenotype (e.g, activation or memory). A non-limiting example of an assay for measuring the quantity and/or characteristics of B cells is flow cytometry. BCRs may also be sequenced, to measure the BCR repertoire and the relative number of B cells expressing a certain BCR (e.g., a preferred BCR).

[0175] Neutralizing capacity of serum or antibody from subjects administered at least one divalent immunogen, multimer, or pharmaceutical composition disclosed herein may be measured by, e.g, a neutralization assay. Neutralization of different viral strains may be measured to assess breadth of neutralization.

[0176] The present disclosure also provides methods of forming a ring structure comprising a divalent immunogen disclosed herein and a BCR or antibody. In some embodiments, the BCR is bound to both antigens or epitopes thereof with 1:1 stoichiometry. In some embodiments, the ring structure is preferentially formed by a certain (e.g., preferred) BCR or antibody. In some embodiments, the certain BCR or antibody is a broadly neutralizing BCR or antibody.

[0177] In some embodiments, the BCR or antibody is in a relaxed state. In some embodiments, the divalent immunogen comprises a specific antigenic spacing that allows the BCR and/or antibody to bind with a Fab-Fab angle of about 20 degrees to about 150 degrees. In some embodiments, the Fab-Fab angle is about 90 degrees to about 140 degrees. In some embodiments, the Fab-Fab angle is about 120 degrees.

[0178] In some embodiments, the ring structure is formed in a subject. In some embodiments, subject is a human.