HIV vaccine immunogen

Abstract

The present disclosure relates to an HIV vaccine immunogen, and specifically, the present disclosure provides an HIV immunogen and its use in producing an immune response in a subject. Methods of isolating anti-HIV antibodies and uses thereof are also disclosed. The disclosure further provides methods of treating or preventing human immunodeficiency virus type 1 (HIV-1) infection in a subject using the disclosed HIV immunogens and / or antibodies.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2019800908563, application date November 27, 2019, and invention name “HIV vaccine immunogen”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 775,192, filed on December 4, 2018. The above application is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on November 14, 2019, named 070413_20403_SL.txt, and is 178,838 bytes in size. Technical Field

[0006] The present disclosure relates to immunogenic polypeptides, and in particular, immunogenic polypeptides capable of stimulating an immune response to the human immunodeficiency virus (HIV). Background Art

[0007] Single cell antibodies cloned from human donors infected with HIV-1 show that broadly neutralizing anti-HIV-1 antibodies (bNAbs) are unique in that they are highly somatically mutated. In addition, a high degree of somatic mutation is essential for binding to natural HIV-1 Env and bNAb neutralizing activity. The aggregation of a large number of mutations suggests that bNAbs evolve to respond to iterations of somatic hypermutations and selection in germinal centers (GCs). As revealed by prospective studies in humans, bNAbs do this to respond to viral escape variants caused by antibody pressure. In summary, these observations suggest that vaccination to elicit bNAbs requires a series of sequential immunogens, starting with immunogens that induce the expansion of B lymphocytes carrying germline precursors of bNAbs.

[0008] The idea that sequential immunization can shepherd bNAb development has been demonstrated by experiments with genetically modified mice carrying inferred germline (iGL) precursors of human bNAbs. However, priming immunogens used to initiate responses fail to activate and expand B cells expressing inferred precursors of bNAbs in animals with polyclonal antibody repertoires. In fact, almost all iGLs of bNAbs are unable to bind to natural-like HIV-1 immunogens or neutralize HIV-1 strains. Therefore, a key goal of HIV-1 vaccine development has been to design immunogens that recruit B cells expressing bNAb precursors to GC responses in animals with polyclonal repertoires, including primates.

[0009] To this end, germline targeting approaches designed for immunogens have focused on producing immunogens that bind to specific bNAb precursors with high affinity, with the rationale that B cell recruitment to GCs depends in part on the affinity of the receptor for the antigen. However, this approach effectively limits the pool of recruited B cells in terms of quality and quantity. Moreover, it cannot explain the following findings: each GC can accommodate a variety of different founder B cells with a wide range of affinities, and GC entry is limited by competition rather than absolute affinity. An alternative approach is to design immunogens that enhance the availability of targeted epitopes while masking off-target sites. This approach differs from germline targeting in that it is unrelated to the affinity of specific germline antibodies for antigens. Instead, it aims to recruit and amplify different groups of precursors specific to the target site. Both approaches aim to produce amplified B cell clones, which can then be enhanced by sequential immunogens to guide bNAb production. So far, none of these methods have been shown to be able to amplify B cell clones specific to the target HIV-1 target in wild-type animals.

[0010] Therefore, there is a pressing need for immunogens that are able to stimulate immune responses to human immunodeficiency virus (HIV), for example by expanding B cell clones specific for desired HIV-1 targets. Summary of the invention

[0011] Various embodiments described in this document address the above unmet needs and / or other needs by providing HIV immunogens and uses thereof.

[0012] In one aspect, the present disclosure relates to an immunogen that stimulates an immune response (e.g., an HIV immune response) in a subject in need thereof. The immunogen comprises a polypeptide having a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 11, and 13. The polypeptide comprises substitutions at positions corresponding to N133, N137, and N156 of SEQ ID NO: 1. In one example, the polypeptide comprises an N156Q substitution or a conservative substitution of N156. In another example, the polypeptide comprises a V134Y, T135A, I138L, T139L, D140S, D141N, T320F, Q328M, T415V substitution, or a conservative substitution thereof.

[0013] The above immunogens are (for example, K D About 50 μM or less) affinity binds to broadly neutralizing antibodies. Examples of broadly neutralizing antibodies may include 10-1074 and PGT121 broadly neutralizing antibodies.

[0014] The isolated nucleic acid encoding the above-mentioned polypeptide, the vector comprising the nucleic acid and the host cell comprising the nucleic acid are also within the scope of the present invention. The host cell can be used in the method for producing the polypeptide. The method includes culturing the host cell in a culture medium under conditions that allow expression of the polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cells or the culture medium of the cells.

[0015] In another aspect, the present disclosure provides a protein complex comprising at least one of the above polypeptides and a viral particle comprising at least one of the above polypeptides.

[0016] In another aspect, the present disclosure provides an immunogenic composition for stimulating an immune response in a subject in need thereof. The immunogenic composition comprises (i) the above-mentioned polypeptide, nucleic acid, host cell, protein complex or viral particle; and (ii) a pharmaceutically acceptable carrier. The method may further include administering the composition two or more times. Administration of the composition may result in an increase in the number of broadly neutralizing antibodies in serum that can recognize the V3-glycan epitope.

[0017] In another aspect, the present disclosure provides a method for stimulating an immune response in a subject in need thereof. The method comprises administering to the subject an effective amount of a composition comprising the above-mentioned polypeptide, nucleic acid, host cell, protein complex or virus-like particle (VLP) or a combination thereof.

[0018] In another aspect, the present disclosure provides a method for treating or preventing HIV infection in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the above-mentioned polypeptide, nucleic acid, host cell, protein complex or viral particle, or a combination thereof. In some embodiments, the method may also comprise administering to the subject a therapeutically effective amount of an antiviral agent.

[0019] In another aspect, the present disclosure provides use of the above polypeptide, nucleic acid, host cell, protein complex or viral particle or a combination thereof in the preparation of a medicament for treating or preventing HIV infection in a subject.

[0020] In another aspect, the present disclosure provides a method for detecting or isolating HIV-1 binding antibodies in a subject infected with HIV-1. The method comprises: (i) providing the above-mentioned polypeptide, nucleic acid, host cell, protein complex or viral particle, or a combination thereof; (ii) contacting the immunogenic composition with a certain amount of body fluid from the subject; and (iii) detecting the binding of HIV-1 binding antibodies to the polypeptide, thereby detecting or isolating HIV-1 binding antibodies in the subject.

[0021] In another aspect, the present disclosure provides a kit for stimulating an immune response in a subject. The kit comprises (i) one or more unit doses of the above-mentioned polypeptide, nucleic acid, host cell, protein complex or viral particle; (ii) instructions for administering the polypeptide, nucleic acid, host cell, protein complex or viral particle; and (iii) optionally, an adjuvant.

[0022] The present disclosure also provides an isolated anti-HIV antibody or antigen binding portion thereof comprising a complementarity determining region having a sequence at least 75% identical to a polypeptide sequence listed in Tables 4, 5, 6, 7, 9, 10 and 11.

[0023] Pharmaceutical compositions comprising the isolated anti-HIV antibodies or antigen-binding portions thereof, and a pharmaceutically acceptable carrier or excipient are also within the scope of the present disclosure.

[0024] In another aspect, the present disclosure provides a method for preventing or treating HIV infection or HIV-related diseases, comprising the steps of: (i) identifying a patient in need of such prevention or treatment, and (ii) administering to the patient a first therapeutic agent comprising a therapeutically effective amount of at least one of the above-mentioned anti-HIV antibodies or antigen-binding portions thereof. The present disclosure further provides a kit comprising a pharmaceutically effective amount of at least one of the above-mentioned isolated anti-HIV antibodies or antigen-binding portions thereof in a pharmaceutically acceptable dosage unit.

[0025] One or more embodiments of the invention are described in detail in the following description. Other features, objects, and advantages of the invention will be apparent from the description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1a , 1b and 1c show the characterization of the RC1 immunogen. Figure 1aThe positions of N-glycans (colored spheres) and the GDIR motif (SEQ ID NO: 15) (red surface) in the V3-glycan patch of wtBG505, 11MUTB and RC1 Env trimers are shown. The coordinates of the glycans are positioned from top to bottom on a surface representation of the wtBG505 Env trimer structure (PDB 5T3Z) (N137 glycan from PDB 5FYL). Figure 1b shows wtBG505 (PDB 5T3Z) (left) and RC1 (right) in complex with 10-1074 Fab ( The Env trimer-Fab complex is shown in the side view as a surface representation, with glycan atoms as colored spheres. The small and medium panels show a close-up overlay of the boxed region of the complex of 10-1074 with wtBG505 and RC1. The protein region is shown as a cartoon representation (10-1074V H and V L Figure 1c shows the SPR binding data (R 2004) from an experiment in which the Fab of the common iGL of PGT121 and 10-1074 was injected into the immobilized Env trimer as shown. eg , equilibrium binding response, relative to the logarithm of the injected protein concentration). NB = no binding above background.

[0027] Figures 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 2k and 21 show that immunization of wild-type mice with RC1 stimulates anti-polysaccharide plaque antibodies. Figure 2a is a representative ELISA result showing the binding of serum from knock-in mice carrying PGT121 / 10-1074iGL antibodies to 11MUTB (left) and RC1 (right) after immunization with 11MUTB (left) and RC1 (right). Controls include unimmunized serum, purified PGT121 and iGL-PGT121. Figure 2b shows the area under the curve (AUC) of the ELISA in Figure 2a, but the results of two independent experiments using three mice each time are combined. Each point represents the serum of one mouse. Figure 2d shows representative ELISA results of the binding of serum from wild-type mice immunized with 11MUTB (left) and RC1 (right) to 11MUTB and RC1, respectively. Figure 2d shows the AUC of the ELISA as shown in c, but the results of 2 independent experiments using 3 mice each time are combined. Each point represents the serum of one mouse. Figure 2e shows the binding of serum from a representative wild-type mouse immunized with RC1 to RC1 and RC1-glycan KO in the ELISA. Figure 2f shows the ratio of the AUC of the RC1 ELISA to the RC1-glycan KO ELISA as shown in Figure 2e. This figure shows the combined results of 7 experiments using 2-3 mice immunized with RC1 each time. Each point represents one mouse. Figure 2g shows representative ELISA results showing the binding of serum from wild-type mice immunized with 11MUTBΔ301 to 11MUTBΔ301. Figure 2h shows the ratio of the AUC of the RC1 ELISA to the RC1-glycan KO ELISA of wild-type mice immunized with RC1 or RC1-4fill. Figure 2i is a pie chart showing the binding of serum from wild-type mice immunized with 11MUTBΔ301 to 11MUTBΔ301. H Figure 2. Clonal expansion of RC1-binding germinal center B cells as determined by gene sequencing. Colored pie slices are proportional to the number of clones associated with them. White represents a single IgV H Sequences. The number in the center indicates the number of heavy chains analyzed. Figure 2j shows the IgH nucleotide mutations of unimmunized mice and mice immunized with RC1 in Figure 2i. Figure 2k shows the binding of monoclonal antibodies obtained from mice immunized with RC1 to RC1 and RC1-glycan KO in ELISA. Figure 2l shows the binding of Ab275 isolated from wild-type mice immunized with RC1 and RC1-4fill by ELISA on the indicated Env proteins. MUR and Ab276 MUR Figure 21 discloses "GAIA" as SEQ ID NO:16.

[0028] Figures 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i and 3j are panels showing that immunization of macaques with RC1-4fill VLPs elicits anti-V3-glycan plaque antibodies similar to iGL bNAbs. Figure 3a shows a representation of RC1-4fill VLPs showing RC1, spytag, spycatcher and VLPs. Figure 3b shows electron micrographs of VLPs (top) and RC1-4fill-VLPs (bottom). Figure 3c shows the binding of sera from 4 rabbits immunized with RC1-4fill VLPs, unimmunized controls and monoclonal antibodies PGT121 and 3BNC117 to RC1 (black) and RC1-glycan KO (grey), shown as the area under the ELISA curve (AUC). Figure 3d shows the binding of sera from 8 rhesus macaques immunized with RC1-4fill VLPs, unimmunized controls, and monoclonal antibodies PGT121 and 3BNC117 to RC1 (black) and RC1-glycan KO (grey), shown as the area under the ELISA curve (AUC). Figure 3e is a representative flow cytometric dot plot showing the binding of macaque germinal center B cells to RC1-PE (Y axis) and RC1-AF647 or RC1-glycan KO (X axis) from unimmunized (left) and immunized (right) macaques. Figure 3f shows the percentage of all B cells in the germinal center of lymph node samples from 4 unimmunized or 4 immunized macaques that bind to RC1 but not to RC1-glycan KO, as determined by flow cytometry. Figure 3g is a pie chart showing the clonal expansion of RC1-binding germinal center B cells determined by IgH gene sequencing. The numbers in the center represent the IgV analyzed. H Figure 3h shows the IgV sequences of all the sequences in Figure 3g. H Mutation, each dot represents an IgV H Figure 3i shows the iGL sequence of CDRL3 of PGT121 / 10-1074 (SEQ ID NO: 446) and a logo plot of all IgL chains from RC1 cloned GC B cells bound to immunized macaque GC B cells. Figure 3j shows a portion of the IgL CDR3 sequence cloned from GC B cells from 4 unimmunized and 4 RC1 immunized macaques showing a DSS-like motif.

[0029] Figures 4a, 4b, 4c, 4d and 4e show binding of monoclonal antibodies from macaques immunized with RC1-4fill VLPs to V3-glycan plaques. Figure 4a shows ELISA results of monoclonal macaque antibodies binding to RC1 and RC1-glycan KO-GAIA ("GAIA" disclosed as SEQ ID NO: 16). Controls were 10-1074 and 3BNC1 17. Figure 4b shows the length of IgH CDR3 of V3-glycan plaque-specific macaque antibodies. Figure 4c shows the length of IgV of V3-glycan plaque-specific macaque antibodies. H and IgV L Figure 4d shows the protein expression of antibody Ab876 NHP 、Ab897 NHP 、Ab933 NHP 、Ab936 NHP 、Ab1170 NHP , 3BNC117 and 10-1074. Figure 4e discloses "GAIA" as SEQ ID NO:16.

[0030] Figures 5a, 5b and 5c show the structural comparison of 10-1074 and the antibodies stimulated to bind to the RC1 immunogen. Figure 5a shows the structural comparison of 10-1074 Fab with other V3-glycan plaque bNAb Fabs (PGT128 and PGT135; PDB 5ACO and 4JM2) (left), Ab275 MUR (Second from left), Ab874 NHP (Third from left) and Ab897 NHP (right) A top view of the binding orientation compared to the Fab structures. The Env and Fab structures are shown as cartoon representations. Figure 5b (upper panel) shows the V3-glycan patch of 10-1074 (left) and the stimulated antibody Fab (right third panel) bound to one monomer (protomer) of the RC1 trimer. H -V L Domains (from 10-1074 (left), Ab275 bound to RC1 Env trimer MUR (Second from left), Ab874 NHP (Third from left) and Ab897 NHP(Right) Cryo-EM structure of the complex). The GDIR residues (SEQ ID NO: 15) on gp120 are highlighted in red and the glycan coordinates are shown as colored spheres. Figure 5b (lower panel) shows a 90° rotation of the complex in the upper panel to show a top view of the antibody binding site with the CDRs highlighted as loops and gp120 glycans (colored spheres) and the region of GDIR (SEQ ID NO: 15) from RC1 (red) positioned on the antibody binding site. Figure 5c shows a comparison of the GDIR motif (SEQ ID NO: 15) with 10-1074 and its interaction with the stimulated antibody.

[0031] Figures 6a and 6b show the characterization of RC1 by evaluating its interaction with bNAbs using ELISA. Figure 6a shows that V1-V2-specific bNAbs that interact with N156 glycan 32 show reduced binding to RC1 compared to BG505, and the lack of N156 PNGS enhances the neutralization of PGT121 and 10-1074. Figure 6b shows that bNAbs targeting the V3-glycan epitope, CD4 binding site, or gp120-gp41 interface bind to RC1 similarly to their binding to BG505.

[0032] Figures 7a, 7b, 7c and 7d show the interaction of RC1 with 10-1074 (Figure 7a), Ab874 NHP (Figure 7b), Ab275 MUR (Figure 7c) and Ab897 NHP (Fig. 7d) Single particle cryo-EM study of RC1 complexed with the antigen-binding fragment (Fab).

[0033] Figure 8 Sera from RC1 immunized mice were shown to cross-react with 11MUTB but not with the more native 10MUT Env or BG505.

[0034] Figures 9a, 9b, 9c, 9d and 9e show the characterization of RC1 and RC1-4fill and their responses to off-target sites.

[0035] Fig.10 Characterization of humoral responses elicited by RC1 and RC1-4fill in wild-type mice is shown. Antibody genes from single GC B cells that bound to RC1 but not to RC1-glycan KO were sequenced.

[0036] Figures 11a, 11b, 11c and 11d show that RC1 and RC1-4fill expand V3-glycan plaque-specific B cells in wild-type mice. MUR) or RC1-4fill(Ab276 MUR ) all bound to 11MUTB, but not to BG505 or the peptide covering the crown of the V3 loop ( FIG11 a ). MUR With a K of about 30 nM D Binds to RC1 (Figure 11b). Importantly, Ab275 MUR Retained binding to 11MUTB (K D The obtained mutation was critical for binding, as reversion to the iGL sequence resulted in loss of binding to RC1 (Fig. 11d).

[0037] Fig.12 VLP-RC1-4fill was shown to elicit V3-glycan plaque antibodies in rabbits and macaques. Sera from macaques primed with RC1-4fill VLPs showed sequential decreases in binding to the more natural-like immunogens 11MUTB and 10MUT.

[0038] Figures 13a, 13b, 13c, 13d, 13e, 13f, 13g, 13h, 13i and 13j (collectively referred to as "Figure 13") are a set of figures showing the characterization of immunogens (including RC1-3fill). Figure 13a is a chart showing the size exclusion chromatography (SEC) traces of RC1, RC1-3fill and RC1-4fill immunogens. Figure 13b provides representative yields of each immunogen expressed in 1L of HEK293T 6E cells. Figures 13c, 13d, 13e and 13f are a set of figures showing SEC curves and electron micrographs of RC1 and RC1-3fill immunogens. Figure 13g shows representative SEC curves for the purification of AP205-RC1-VLP and AP205-RC1-3fill-VLP. Figure 13d shows electron micrographs of AP205-RC1-VLP (left) and AP205-RC1-3fill-VLP (right). Figure 13e shows representative SEC curves for the purification of mi3-RC1-NP and mi3-RC1-3fill-NP. Figure 13f shows electron micrographs of mi3-RC1-NP (left) and mi3-RC1-3fill-NP (right). Figures 13f and 13h show the initial purification of both AP205-RC1-VLP (Figure 13f) and mi3-RC1-NP (Figure 13h), as well as the SEC curves of the reinjected samples at 28 days (AP205) and 11 days (mi3). Figures 13i and 13j show the binding of sera from 6 WT mice immunized with mi3-RC1-NP (Figure 13i) or mi3-RC1-3fill-NP (Figure 13j), unimmunized controls, and monoclonal antibodies 10-1074 and 3BNC117 to RC1 and RC1-glycan KO, shown as the area under the ELISA curve (AUC). DETAILED DESCRIPTION

[0039] The disclosed immunogens that stimulate an immune response in a subject are based on the unexpected discovery that the novel immunogen RC1 and its variants activate B cells that express precursors of bNAbs in a polyclonal pool.

[0040] Broadly neutralizing antibodies (bNAbs) protect against HIV-1 infection, suggesting that vaccines that elicit them will be effective. However, one of the major obstacles is that vaccination does not elicit bNAbs, in part because B cells expressing germline bNAb precursors do not respond to native-like HIV-1 envelope (Env) antigens. Therefore, the present disclosure provides immunogens that promote recognition of V3-glycan patches on HIV-1 Env while masking non-conserved immunodominant regions (e.g., by adding glycans and / or polymerizing on virus-like particles). The present disclosure demonstrates that immunization of mice, rabbits, and rhesus monkeys with the disclosed immunogens (e.g., RC1, RC1-4fill, RC1-3fill) elicits serological responses targeting V3-glycan patches. In addition, antibody clones and cryo-EM structures of antibody-Env complexes confirmed that RC1 immunization expanded B cell clones carrying anti-V3-glycan patch antibodies similar to the predicted precursors of human bNAbs. Thus, the disclosed immunogens, such as RC1, are suitable priming immunogens for use in a sequential vaccination strategy to stimulate an immune response (eg, an HIV immune response) in a subject.

[0041] I. Immunogens and immunogenic compositions

[0042] A. Peptide

[0043] The present disclosure provides an immunogen and variants thereof that stimulate an immune response (e.g., HIV immune response) of a subject in need thereof. In some embodiments, the immunogen comprises a portion of an HIV envelope protein located on the surface of HIV, i.e., gp120. Gp120 is the N-terminal segment of the HIV envelope protein gp160 anchored in a double membrane at its carboxyl terminal region. Gp120 extends into the water environment around the virion, while its C-terminal counterpart gp41 spans the membrane. The gp120 molecule consists of a 60,000 dalton polypeptide core, which is extensively modified by N-linked glycosylation to increase the apparent molecular weight of the molecule to 120,000 daltons. The amino acid sequence of gp120 contains five relatively conserved domains, interspersed with five hypervariable domains. The positions of 18 cysteine ​​residues in the gp120 primary sequence and 13 of the approximately 24 N-linked glycosylation sites in the gp120 sequence are common among all gp120 sequences.

[0044] In some embodiments, the immunogen may comprise the Env V3 region of gp120. The Env V3 region of gp120 encompasses the V3-glycan patch epitope, which comprises a set of high mannose and complex N-glycans surrounding the Env V3 region. In the V3-glycan patch epitope, glycosylation typically occurs at gp120 residues N133, N137, N156, N295, N301, N332, N339, N385, and N392. bNAbs, such as PGT121, 10-1074, and BG18, target the V3-glycan patch epitope. They use an extended CDRH3 loop and portions of CDRL1 and CDRL3 to traverse the glycans to contact the highly conserved GDIR motif (G324-D325-I326-R327) (SEQ ID NO: 15) located at the base of the V3 loop.

[0045] In some embodiments, the immunogen may comprise one or more modifications in the Env V3 region of gp120. The immunogen comprises a polypeptide having a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 11, and 13 listed in Table 1. The polypeptide may comprise a substitution of one or more glycosylation sites (e.g., N133, N137, N156, N295, N301, N332, N339, N385, and N392) in the Env region. For example, the polypeptide may comprise a substitution at a position corresponding to N133, N137, and N156 of SEQ ID NO: 1. In one example, the polypeptide comprises an N156Q substitution or a conservative substitution of N156. In another example, a polypeptide, such as RC1 (SEQ ID NO: 2; Table 1), comprises deletions at N133, N137, and N156, and other substitutions, including V134Y, T135A, 1138L, T139L, D140S, D141N, T320F, Q328M, and T415V. As will be demonstrated in the Examples, the disclosed immunogens, such as RC1, activate and expand a variety of B cells expressing antibodies similar to human V3-glycan bNAb precursors in mice, rabbits, and macaques.

[0046] Table 1: Sequences of HIV immunogens

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] The amino acid or nucleic acid sequence of the above HIV immunogens contains a secretory leader sequence at the N-terminus (for amino acid sequences) or at the 5' terminus (for nucleic acid sequences). The secretory leader sequence is a general secretion signal and is not part of the final / mature expressed protein. As will be appreciated by those of ordinary skill in the art, other secretory leader sequences may also be used to produce the same final / mature HIV immunogen.

[0059] Table 2: Immunogen variants and specificity modifications

[0060]

[0061]

[0062]

[0063] "Polypeptide" is used in its conventional sense, i.e., to refer to a sequence of amino acids. Polypeptides are not limited to products of a specific length. Peptides, polypeptides, and proteins are all included in the definition of polypeptides, and such terms are used interchangeably herein unless otherwise expressly stated. This term also includes post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art that are naturally occurring and non-naturally occurring. A polypeptide may be a complete protein or a subsequence thereof. The term "variant" of a polypeptide, as used herein, is a polypeptide that is generally different from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring, or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences disclosed herein and evaluating one or more biological activities of the polypeptides described herein and / or using any of some techniques known in the art.

[0064] For example, in a protein structure, certain amino acids can replace other amino acids without significantly losing their ability to bind to other polypeptides (e.g., antigens) or cells. Since the biological functional activity of a protein is determined by the binding ability and properties of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, accordingly, in its underlying DNA coding sequence to obtain a protein with similar properties. Therefore, it is expected that various changes can be made to the peptide sequence of the disclosed composition or the corresponding DNA sequence encoding the peptide without significantly losing its biological use or activity.

[0065] Variant sequences include those sequences in which conservative substitutions have been introduced by modifying the polynucleotides encoding the polypeptides disclosed herein. Amino acids can be classified according to physical properties and contributions to secondary and tertiary protein structures. Such conservative modifications include substitutions, additions and deletions of amino acids. Conservative amino acid substitutions are amino acid substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0066] "Sequence identity" or "homology" refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that are identical to the non-variant sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent homology. In specific embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.

[0067] The polypeptide variant sequence may have 70% or higher (i.e. 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequence described in the present disclosure. Polypeptide variants may also include polypeptide fragments comprising continuous segments of the amino acid sequences disclosed herein of various lengths. Polypeptide variant sequences comprise at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more continuous peptides of one or more sequences disclosed herein, and all intermediate lengths therebetween.

[0068] The above immunogens can specifically bind to bNAbs. bNAbs are neutralizing antibodies that neutralize multiple HIV-1 strains. bNAbs are unique in that they target conserved epitopes of the virus. Examples of broadly neutralizing antibodies may include, but are not limited to, VRC26.25, PCT64-24E, VRC38.01, PG9, PGDM1400, CHO1, BG18, DH270.1, DH270.6, PGDM12, VRC41.01, PGDM21, PCDN-33A, BF520.1, VRC29.03, PGT121, 10-1074, N49-P7, N6, NC-Cow1, IOMA, CH235, CH235.12, b12, VRC01, 3BNC117, CH103, VRC-PG05, VRC34.01, ACS202, PGT151, 35022, 8ANC195, DH511.11P. Among these bNAbs, BG18, DH270.1, DH270.6, PGDM12, VRC41.01, PGDM21, PCDN-33A, BF520.1, VRC29.03, PGT121, 10-1074 broadly neutralizing antibodies specifically bind to V3 glycans. In some embodiments, the disclosed immunogens have a dissociation constant (K D ) binds to PGT121 or 10-1074 broadly neutralizing antibodies with an affinity of about 50 μM or less.

[0069] The terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen and not to other antigens. -6 M, for example, less than about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K D ) binding, when measured by, for example, ELISA, equilibrium dialysis or 2000 surface plasmon resonance (SPR) technology, or Scatchard analysis of antibody binding to antigen-positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least twice as high as its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein).

[0070] In another aspect, the disclosure provides immunogenic polypeptides polymerized on virus-like particles (VLP) (for example, retrovirus-like particles, HIV-like particles). In the context of the present disclosure, virus-like particles or retrovirus-like particles are structures surrounded by membranes, which include viral envelope proteins embedded in the membranes of host cells producing them therein, and preferably, other viral core proteins in VLP. These VLPs do not contain complete viral nucleic acids, and they are non-infectious. Ideally, there are enough envelope proteins on the surface of VLP so that when the VLP preparation is formulated into immunogenic compositions and administered to animals or people, immune responses (cell-mediated or humoral) can be produced. Ideally, compared with naturally occurring viral envelope proteins, Env proteins are truncated from the carboxyl terminal. In the context of the present invention, "truncated" envelope proteins are proteins containing less than full-length cytoplasmic domains, but they retain surface antigenic determinants that are targeted to produce immune responses, preferably protective immune responses, and they retain enough envelope sequences for suitable precursor processing and film insertion. The skilled artisan can use recombinant DNA technology and viral coding sequences readily available to the public to produce truncated viral envelope proteins. For example, the coding sequence of the viral envelope protein can be engineered for expression in a baculovirus expression vector, such as using a commercially available baculovirus vector, under the control of a viral promoter, the sequence is appropriately modified to achieve functional connection of the coding sequence with a regulatory sequence, and a portion of the coding sequence encoding the cytoplasmic domain of the envelope protein is truncated (deleted), still by using an appropriate translation termination signal and a sequence that allows the truncated envelope and related fragments to be effectively spliced ​​into the vector. A specific exemplary truncated SIV envelope protein lacks the 89 amino acids located at the carboxyl terminus of the naturally occurring SIV envelope protein.

[0071] In another aspect, the present disclosure provides a protein complex comprising at least one of the above-mentioned immunogenic polypeptides that are polymerized via covalent or non-covalent bonding / interactions (e.g., van der Waals interactions). For example, two or more immunogenic polypeptides can be cross-linked by one or more cross-linking agents. A cross-linking agent is a reagent having a terminal end that can react with a specific functional group (e.g., a primary amine or sulfhydryl) on a protein or other molecule. A cross-linking agent can connect two or more molecules via covalent bonds. Cross-linking agents include, but are not limited to, amine-amine cross-linking agents (e.g., disuccinimidyl suberate (DSS)), amine-thiol cross-linking agents (e.g., N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS)), carboxyl-amine cross-linking agents (e.g., dicyclohexylcarbodiimide (DCC)), thiol-carbohydrate cross-linking agents (e.g., N-β-maleimidopropionic acid hydrazide (BMPH)), thiol-thiol cross-linking agents (e.g., 1,4-bismaleimidobutane (BMB)), photoreactive cross-linking agents (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS)), and chemoselective ligation cross-linking agents (e.g., NHS-PEG4-azide).

[0072] B. Nucleic Acids

[0073] Another aspect of the present disclosure is characterized by an isolated nucleic acid comprising a sequence encoding the above-mentioned polypeptide or protein. Nucleic acid refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), or a DNA or RNA analog. DNA or RNA analogs can be synthesized from nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0074] "Isolated nucleic acid" refers to a nucleic acid whose structure is not identical to the structure of any naturally occurring nucleic acid or any fragment of a naturally occurring genomic nucleic acid. Thus, the term encompasses, for example, (a) a DNA having a portion of the sequence of a naturally occurring genomic DNA molecule, but not flanked by two coding sequences that flank that portion of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or genomic DNA of a prokaryotic or eukaryotic organism in such a way that the resulting molecule is different from any naturally occurring vector or genomic DNA; (c) a separate molecule, such as a cDNA, a genomic fragment, a fragment generated by the polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The above nucleic acids can be used to express polypeptides, fusion proteins or antibodies of the present invention. For this purpose, the nucleic acid can be operatively linked to a suitable regulatory sequence to produce an expression vector.

[0075] The nucleic acid and amino acid sequences disclosed herein are shown using standard letter abbreviations for nucleotide bases and the single letter code for amino acids. Only one strand is shown for each nucleic acid sequence, but the complementary strand is understood to be included in the displayed strand by any reference.

[0076] The present disclosure also includes vectors containing the coding sequences of the disclosed immunogens, host cells containing the vectors, and methods for preparing substantially pure immunogens, comprising the steps of introducing the coding sequences of the immunogens into host cells and culturing the host cells under appropriate conditions to produce and secrete the immunogens. The immunogens so produced can be harvested in a conventional manner. Therefore, the present invention also relates to methods of expressing the immunogens and biological equivalents disclosed herein, assays using these gene products, and recombinant host cells containing DNA constructs expressing these receptor proteins.

[0077] The disclosed immunogens can be recombinantly expressed by cloning a nucleic acid molecule encoding the immunogen into an expression vector (e.g., pcDNA3.neo, pcDNA3.1, pCR2.1, pBlueBacHis2, or pLITMUS28) containing a suitable promoter and other suitable transcriptional regulatory elements, and transferred into a prokaryotic or eukaryotic host cell to produce the immunogen. Techniques for such manipulations are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, which are well known and readily available to those of ordinary skill in the art. Therefore, another aspect of the present invention includes host cells that have been engineered to contain and / or express a DNA sequence encoding an immunogen. Such recombinant host cells can be cultured under appropriate conditions to produce the disclosed immunogens or biologically equivalent forms. Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as Escherichia coli (E. coli), fungal cells such as yeast, mammalian cells, including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells, including but not limited to cell lines derived from Drosophila and silkworm.

[0078] For example, one insect expression system utilizes Spodopterafrugiperda (Sf21) insect cells (Invitrogen) and a baculovirus expression vector (pAcG2T, Pharmingen). In addition, mammalian species that may be suitable and are commercially available include, but are not limited to, L cell LM (TK-) (ATCC CCL 1.3), L cell LM (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL61), 3T3 (ATCC CCL92), NIH / 3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL26), MRC-5 (ATCC CCL 171), and CPAE (ATCC CCL 209).

[0079] A variety of mammalian expression vectors can be used to express recombinant immunogens in mammalian cells. Expression vectors are defined herein as DNA sequences required for transcribing cloned DNA and translating its mRNA in a suitable host. Such vectors can be used to express eukaryotic DNA in a variety of hosts, such as bacteria, blue-green algae, plant cells, insect cells, and animal cells. Specially designed vectors can shuttle DNA between hosts such as bacteria-yeast or bacteria-animal cells. Construction of suitable expression vectors should contain: a replication origin for autonomous replication in host cells, a selectable marker, a limited number of useful restriction enzyme sites, a potential for high copy number, and an active promoter. Promoters are defined as DNA sequences that guide RNA polymerase to bind DNA and initiate RNA synthesis. A strong promoter is a promoter that causes mRNA to be initiated at a high frequency.

[0080] Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids or viruses. Commercially available mammalian expression vectors suitable for immunogen expression include, but are not limited to, pIRES-hyg (Clontech), pIRES-puro (Clontech), pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).

[0081] A variety of bacterial expression vectors can also be used to express the disclosed immunogens in bacterial cells. Commercially available bacterial expression vectors that may be suitable for immunogen expression include, but are not limited to, pCR2.1 (Invitrogen), pET11a (Novagen), λgt11 (Invitrogen), and pKK223-3 (Pharmacia).

[0082] In addition, a variety of fungal cell expression vectors can be used to express immunogens in fungal cells. Commercially available fungal cell expression vectors that can be used to express recombinant immunogens include, but are not limited to, pYES2 (Invitrogen) and Pichia pastoris expression vector (Invitrogen).

[0083] In addition, a variety of insect cell expression vectors can be used to express recombinant receptors in insect cells. Commercially available insect cell expression vectors suitable for recombinant expression of immunogens include, but are not limited to, pBlueBacIII and pBlueBacHIs2 (Invitrogen) and pAcG2T (Pharmingen).

[0084] The expression vector can be introduced into the host cell by any of a variety of techniques including, but not limited to, transformation, transfection, protoplast fusion, and electroporation. Transformation is meant to cover genetic changes in the target cell caused by the incorporation of DNA. Transfection is meant to include any method known in the art for introducing an immunogen into a test cell. For example, transfection includes calcium phosphate or calcium chloride-mediated transfection, lipofection, electroporation, and infection using, for example, a viral vector such as a recombinant retroviral vector containing a nucleotide sequence encoding an immunogen, and a combination thereof. Cells containing expression vectors are analyzed separately to determine whether they produce immunogens. Identification of cells expressing immunogens can be performed by a variety of means, including but not limited to immunoreactivity with specific bNAbs, binding of labeled ligands, and the presence of host cell-related activities regarding immunogens.

[0085] Host cells containing the above nucleic acids are also within the scope of the present invention. Examples include bacterial cells (e.g., E. coli cells, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. See, e.g., Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. In order to produce the polypeptide of the present invention, host cells can be cultured in a culture medium under conditions that allow expression of the polypeptide encoded by the nucleic acid of the present invention, and the polypeptide can be purified from the cultured cells or the culture medium of the cells. Alternatively, the nucleic acid of the present invention can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and a T7 polymerase.

[0086] C. Composition

[0087] In another aspect, the present disclosure provides an immunogenic composition for stimulating an immune response in a subject in need thereof. The immunogenic composition comprises (i) the above-mentioned immunogen, nucleic acid, host cell, protein complex or viral particle; and (ii) a pharmaceutically acceptable carrier. The method may further comprise administering the composition two or more times. Administration of the composition may result in an increase in the number of broadly neutralizing antibodies in the serum that can recognize the V3-glycan epitope.

[0088] An immunogenic composition is a composition comprising an immunogenic peptide that induces a measurable CTL response against a virus expressing the immunogenic peptide, or induces a measurable B cell response (e.g., production of antibodies) against the immunogenic peptide. In one example, an "immunogenic composition" is a composition comprising a disclosed immunogen derived from gp120 or an antigenic fragment thereof. It further refers to an isolated nucleic acid encoding an immunogen, such as a nucleic acid that can be used to express an immunogen (and thus to stimulate an immune response against such a polypeptide).

[0089] For in vitro use, the immunogenic composition can be composed of isolated proteins, peptide epitopes, or nucleic acids encoding proteins or peptide epitopes. For in vivo use, the immunogenic composition will generally comprise a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and / or other reagents. Any specific peptide, such as a disclosed immunogen, or a nucleic acid encoding the immunogen, can be easily tested for its ability to induce CTL or B cell responses by art-recognized assays. The immunogenic composition may comprise an adjuvant well known to those skilled in the art.

[0090] Sterile injectable compositions can be solutions or suspensions in nontoxic parenteral acceptable diluents or solvents. Such solutions include but are not limited to 1,3-butylene glycol, mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, conventional fixed oils are used as solvents or suspension media (e.g., synthetic monoglycerides or diglycerides). Fatty acids, such as but not limited to oleic acid and its glyceride derivatives, can be used to prepare injections, such as natural pharmaceutically acceptable oils, such as but not limited to olive oil or castor oil, its polyoxyethylene form. These oil solutions or suspensions may also include long-chain alcohol diluents or dispersants, such as but not limited to carboxymethyl cellulose or similar dispersants. For formulation purposes, other commonly used surfactants may also be used, such as but not limited to TWEEN or SPANS or other similar emulsifiers or bioavailability enhancers, which are commonly used to prepare pharmaceutically acceptable solids, liquids or other dosage forms.

[0091] Compositions for oral administration can be any orally acceptable dosage form, including capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For tablets, conventional carriers include, but are not limited to, lactose and corn starch. Lubricants are also usually added, such as, but not limited to, magnesium stearate. For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dried corn starch. When an aqueous suspension or emulsion is administered orally, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier or suspending agent. If necessary, certain sweeteners, flavorings or coloring agents can be added.

[0092] The pharmaceutical composition for topical application according to the invention can be formulated as a solution, ointment, emulsion, suspension, lotion, powder, paste, gel, spray, aerosol or oil. Alternatively, the topical preparation can be in the form of a patch or dressing soaked with active ingredients, which can optionally include one or more excipients or diluents. In some preferred embodiments, the topical preparation includes a material that enhances the absorption or penetration of the active agent through the skin or other affected areas. Topical compositions can be used to treat inflammatory diseases of the skin, including but not limited to eczema, acne, rosacea, psoriasis, contact dermatitis and reactions to poison ivy.

[0093] Topical compositions contain a dermatologically acceptable carrier in a safe and effective amount suitable for the skin. "Cosmetically acceptable" or "dermatologically acceptable" compositions or components refer to compositions or components suitable for contact with human skin without excessive toxicity, incompatibility, instability, allergic reactions, etc. The carrier enables the active agent and optional components to be delivered to the skin at a suitable concentration. Therefore, the carrier can be used as a diluent, dispersant, solvent, etc. to ensure that the active substance is applied at a suitable concentration and evenly distributed on the selected target. The carrier can be solid, semi-solid or liquid. The carrier can be in the form of a lotion, emulsion or gel, especially in a form with sufficient thickness or yield point to prevent the active substance from depositing. The carrier can be inert or have dermatological benefits. It should also be physically and chemically compatible with the active ingredients described herein, and should not excessively impair stability, efficacy or other use benefits associated with the composition. Topical compositions can be cosmetic or dermatological products in the form of topical or transdermal applications known in the art, including solutions, aerosols, emulsions, gels, patches, ointments, lotions or foams.

[0094] Pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. "Pharmaceutically acceptable carriers" do not cause undesirable physiological effects after being administered to or on a subject. The carrier in the pharmaceutical composition must be "acceptable" and also means compatible with the active ingredient and capable of stabilizing it. One or more solubilizing agents can be used as drug carriers to deliver active agents. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives and diluents to achieve compositions that can be used as dosage forms. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents and the necessity of using them are described in Remington's Pharmaceutical Sciences. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The therapeutic compound may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects (see, eg, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19).

[0095] The host cells provided in the immunogenic composition may be inactivated or chemically / genetically attenuated bacterial vaccines that do not elicit the cytotoxic T lymphocyte (CTL) immune response necessary for lysis of tumor cells and cells infected with intracellular pathogens.

[0096] II. Methods of Stimulating Immune Responses Using the Disclosed Immunogens

[0097] The immunogens disclosed herein, nucleic acid molecules encoding the disclosed immunogens, host cells, protein complexes or viral particles can be administered to a subject to generate an immune response against a pathogen such as HIV. In another aspect, the present disclosure provides a method for treating or preventing HIV infection in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the above-mentioned immunogen, nucleic acid, host cell, protein complex or viral particle, or a combination thereof. The present disclosure also provides the use of the above-mentioned immunogen, nucleic acid, host cell, protein complex or viral particle or a combination thereof in the preparation of a medicament for treating or preventing HIV infection in a subject.

[0098] In an exemplary application, the composition is administered to a subject having HIV infection or at risk of HIV infection. In other applications, the immunogens disclosed herein can be administered prophylactically, for example, as part of an immunization regimen.

[0099] The immunogen is administered in an amount sufficient to generate an immune response against the HIV virus. Administration causes a sufficient immune response to treat pathogenic infection, such as inhibiting infection and / or reducing signs and / or symptoms of infection. The amount effective for this purpose will depend on the severity of the disease, the general condition of the subject's health, and the robustness of the subject's immune system. The therapeutically effective amount of an immunogen refers to providing subjective relief of symptoms or objectively identifiable improvements as indicated by a physician or other qualified observer.

[0100] Therapeutically effective amount or effective amount refers to the symptom and / or root cause that is enough to prevent, treat (including preventive treatment), alleviate and / or improve any one kind of illness or disease, for example, the medicament of preventing, suppressing and / or treating HIV, for example, the amount of nucleic acid vaccine or other therapeutic agent.In some embodiments, "effective amount" is enough to alleviate or eliminate the symptom of disease such as AIDS.For example, this can be the amount required for suppressing viral replication or measurably changing the external symptoms of viral infection (for example, T cell count increases in the case of HIV-1 infection).Usually, this amount will be enough to measurably suppress the replication or infectivity of virus (for example, HIV).When being administered to a subject, it will be common to use the dosage that will realize the target tissue concentration (for example, in lymphocyte) that has been shown to realize the in vitro inhibition of viral replication.

[0101] The immunogen can be administered by any means known to those skilled in the art (see Banga, A., "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995), locally or systemically, for example, by intramuscular, subcutaneous or intravenous injection, even oral, nasal or anal administration is contemplated. In one embodiment, administration is by subcutaneous or intramuscular injection. In order to extend the time that the disclosed immunogen is available for stimulating a response, the immunogen can be provided as an implant, an oily injection, or a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or a similar particle (see, e.g., Banga, supra). Synthetic polymer-based particulate carriers have been shown to act as adjuvants to enhance immune responses in addition to providing controlled release. Aluminum salts can also be used as adjuvants to generate an immune response.

[0102] Optionally, one or more cytokines such as interleukin (IL)-2, IL-6, IL-12, IL-15, RANTES, granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-a, interferon (IFN)-a or IFN-γ, one or more growth factors such as GM-CSF or G-CSF, one or more co-stimulatory molecules such as ICAM-1, LFA-3, CD72, B7-1, B7-2 or other B7-related molecules; one or more molecules such as OX-40L or 41BBL, or a combination of these molecules can be used as a biological adjuvant (see, for example, Salgaller et al., 1998, J. Surg. Onc01. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cell Biology, 14(6):124-13; Cao et al., 1998, Stem Cell Biology, 14(6):125-13; Cao et al., 1998, Stem Cell Biology, 14(6):126-13; Cao et al., 1998, Stem Cell Biology, 14(6):127-13; Cao et al., 1998, Stem Cell Biology, 14(6):128-13; Cao et al., 1998, Stem Cell Biology, 14(6):129-24; Cao et al., 1998, Stem Cell Biology, 14(6):129-24. Cells16 (Suppl1J.-251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered to the host systemically (or locally). In some examples, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B.7-2, OX-40L, 41BBL and ICAM-1 are administered.

[0103] A pharmaceutical composition comprising an isolated immunogen is provided. In some embodiments, the immunogen is mixed with an adjuvant containing two or more stabilizing detergents, micelle formers, and oils. Suitable stabilizing detergents, micelle formers, and oils are described in detail in U.S. Pat. No. 5,585,103; U.S. Pat. No. 5,709,860; U.S. Pat. No. 5,270,202, and U.S. Pat. No. 5,695,770. A stabilizing detergent is any detergent that keeps the emulsion components as a stable emulsion. Such detergents include polysorbate 80 (TWEEN) (sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured by ICI Americas, Wilmington, DE), TWEEN 40 TM 、TWEEN 20 TM 、TWEEN 60 TM ,, ZWITTERGENTTM3-12, TEEPOL HB7 TM and SPAN 85 TM. These detergents are typically provided in an amount of about 0.05% to 0.5%, such as about 0.2%. Micellar formers are agents that stabilize emulsions formed with other ingredients to form micelle-like structures. Such agents typically cause some irritation at the injection site to recruit macrophages to enhance the cellular response. Examples of such agents include polymeric surfactants described by: BASF Wyandotte publications, for example, Schmolka, J. Am. Oil. Chem. Soc. 54: 110, 1977, and Hunter et al., J. Immunol 129: 1244, 1981, PLURONIC TM L62LF, L101 and L64, PEG1000 and TETRONIC TM 1501, 150R1, 701, 901, 1301 and 130R1. The chemical structures of such agents are well known in the art. In one embodiment, the agent is selected to have a hydrophile-lipophile balance (HLB) between 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be provided in an effective amount, for example, between 0.5 and 10%, or between 1.25 and 5%.

[0104] Controlled release parenteral formulations can be made into implants, oily injections or particle systems. For a broad overview of protein delivery systems, see Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, 1995. Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain therapeutic proteins as a central core. In the microspheres, the therapeutic agent is dispersed throughout the particles. Particles, microspheres and microcapsules less than about 1 μm are usually referred to as nanoparticles, nanospheres and nanocapsules, respectively. The diameter of the capillary is about 5 μm, so only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, 1992).

[0105] Polymers can be used for controlled release of ions. Various degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 53', 1993). For example, the block copolymer poloxamer 407 exists as a viscous but flowable liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective carrier for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425, 1992; and Pec, / . Parent. Sci. Tech. 44 (2): 58, 1990). In addition, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215, 1994). In another aspect, liposomes are used for controlled release of lipid-encapsulated drugs as well as drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Many other systems for the controlled delivery of therapeutic proteins are known (e.g., U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; and U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S. Pat. No. 5,534,496).

[0106] In another embodiment, a pharmaceutical composition includes a nucleic acid encoding a disclosed immunogen. A therapeutically effective amount of the nucleic acid can be administered to a subject to generate an immune response. In a specific non-limiting example, a therapeutically effective amount of a nucleic acid encoding a disclosed gp120 immunogen or an immunogenic fragment thereof is administered to a subject to treat, prevent, or inhibit HIV infection.

[0107] Optionally, one or more cytokines such as IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF-a or IFN-γ, one or more growth factors such as GM-CSF or G-CSF, one or more co-stimulatory molecules such as ICAM-1, LFA-3, CD72, B7-1, B7-2 or other B7-related molecules; one or more molecules such as OX-40L or 41BBL, or a combination of these molecules can be used as a biological adjuvant (see, e.g., Salgaller et al., 1998, J. Surg. Oncol. 68(2): 122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl 1): S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1): 251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465: 381-90). These molecules can be administered systemically to the host. It should be noted that these molecules can be co-administered by inserting nucleic acids encoding the molecules into vectors such as recombinant poxvirus vectors (see, e.g., U.S. Pat. No. 6,045,802). In various embodiments, nucleic acids encoding biological adjuvants can be cloned into the same vector as the disclosed sequences encoding immunogens, or nucleic acids can be cloned into one or more separate vectors for co-administration. In addition, non-specific immunomodulators such as Bacillus Cahnette-Guerin (BCG) and levamisole can be co-administered. One method of administering nucleic acids is direct immunization with plasmid DNA, such as a mammalian expression plasmid. As described above, the nucleotide sequence encoding the disclosed immunogen can be placed under the control of a promoter to increase the expression of the molecule.

[0108] Immunization by nucleic acid constructs is well known in the art and is taught, for example, in U.S. Pat. No. 5,643,578, which describes a method for immunizing vertebrates by introducing DNA encoding a desired immunogen to stimulate a cell-mediated or humoral response, and U.S. Pat. No. 5,593,972 and U.S. Pat. No. 5,817,637, which describe operatively linking a nucleic acid sequence encoding an antigen to a regulatory sequence that enables its expression. U.S. Pat. No. 5,880,103 describes several methods for delivering nucleic acids encoding immunogenic peptides or other antigens to an organism. The method includes delivering the nucleic acid (or its synthetic peptide itself) in liposomes, and an immunostimulatory construct or ISCOMSTM, i.e., a mixture of cholesterol and QuilA. TM Negatively charged cage structures of 30-40 nm in size spontaneously formed on saponin. TMAs a delivery vehicle for antigens, protective immunity has been generated in a variety of experimental infection models, including toxoplasmosis and Epstein-Barr virus-induced tumors (Mowat and Donachie, Immunol. Today 12:383, 1991). TM As low as 1 μg dose of antigen in can generate class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

[0109] In another method of immunization using nucleic acids, the disclosed immunogens can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia viruses, adeno-associated viruses (AAV), herpes viruses, retroviruses, cytomegaloviruses or other viral vectors can be used to express peptides or proteins to stimulate CTL responses. For example, U.S. Pat. No. 4,722,848 describes vaccinia vectors and methods that can be used in immunization protocols. BCG (Bacillus Calmette Guerin) provides another vector for expressing peptides (see Stover, Nature 351: 456-460, 1991).

[0110] In one embodiment, the nucleic acid encoding the disclosed immunogen is introduced directly into the cell. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad's HELIOS™ gene gun. The nucleic acid can be "naked" and consist of a plasmid controlled by a strong promoter. Typically, the DNA is injected into the muscle, although it can also be injected directly into other sites, including tissues near metastases. The injection dose is typically about 0.5 g / kg to about 50 mg / kg, and typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0111] The composition is administered in a single or multiple administrations, depending on the dose and frequency required and tolerated by the subject. In one embodiment, the dose is administered in a single bolus, but in another embodiment, it may be administered periodically until a therapeutic result is achieved. Typically, the dose is sufficient to treat or improve the symptoms or signs of the disease without causing unacceptable toxicity to the subject. Systemic or local administration may be used.

[0112] It may be advantageous to administer the immunogenic compositions disclosed herein together with other agents such as proteins, peptides, antibodies and other antiviral agents such as anti-HIV agents. Examples of such anti-HIV therapeutic agents include nucleoside reverse transcriptase inhibitors such as abacavir, AZT, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, etc., non-nucleoside reverse transcriptase inhibitors such as delavirdine, efavirenz, etc. z), nevirapine, protease inhibitors such as amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, fosamprenavir, ritonavir, saquinavir, tipranavir, etc., and fusion protein inhibitors such as enfuvirtide, etc. In certain embodiments, the immunogenic composition is administered simultaneously with other anti-HIV therapeutic agents. In some examples, the disclosed immunogens are administered together with helper T cells, such as exogenous helper T cells. Exemplary methods for producing and administering helper T cells can be found in International Patent Publication WO 03 / 020904, which is incorporated herein by reference. In certain embodiments, the immunogenic composition is administered sequentially with other anti-HIV therapeutic agents, such as before or after other agents. One of ordinary skill in the art will appreciate that sequential administration can mean administration immediately thereafter, or administration after an appropriate period of time, such as hours, days, weeks, months or even years.

[0113] The disclosed gp120 immunogens or immunogenic fragments thereof and nucleic acids encoding these immunogens can be used in a multi-step immunization program. In some examples, the program includes administering to a subject a therapeutically effective amount of a first immunogen or immunogenic fragment thereof disclosed herein (prime) and boosting the immunogenic response with one or more additional immunogens or immunogenic fragments thereof after an appropriate period of time. The method of stimulating such an immune response is known as "prime-boost". In this method, the antibody response to a selected immunogenic surface is focused by giving the subject's immune system a chance to "see" the antigenic surface in multiple situations. In other words, the use of multiple immunogens or immunogenic fragments thereof having a common antigenic surface selects antibodies that bind to the common immunogenic surface.

[0114] In some examples, immunogens or immunogenic fragments thereof and nucleic acids encoding these immunogens can be administered in a "prime-boost" immunization schedule. For example, immunogens or immunogenic fragments thereof and nucleic acids encoding these immunogens can be administered to a subject before, during, or after administration of a stable gpl40 trimer (see, e.g., Yang et al. J Virol. 76(9): 4634-42, 2002).

[0115] It is also possible to use a mixture containing disclosed immunogenic agents, such as the above-mentioned immunogen, nucleic acid encoding the immunogen, host cells, protein complexes or viral particles or a combination thereof to perform initial immunization, and then use a trimer from a variety of different HIV strains or a trimer as a mixture of a variety of HIV strains to strengthen. Initial immunization can be administered in a single dose or multiple doses, for example, two, three, four, five, six or more doses can be administered to a subject in a few days, weeks or months. Strengthening can be administered in a single dose or multiple doses, for example, two to six or more doses can be administered to a subject in a day, a week or months. Multiple reinforcements can also be given, for example, one to five times, or more times. Different doses can be used for a series of sequential inoculations. For example, a relatively large dose is used in the initial inoculation, and then a relatively small dose is strengthened. The immune response on the surface of the selected antigen can be produced by inoculating a subject once or multiple times with the immunogenic composition disclosed herein.

[0116] III. Immunodiagnostic reagents and kits

[0117] The present disclosure provides a method for detecting or separating HIV-1 binding antibodies in a subject infected with HIV-1. The method includes contacting a sample from the subject, such as but not limited to blood, serum, plasma, urine or sputum samples from the subject with one or more disclosed immunogenic agents, such as the above-mentioned immunogens, nucleic acids encoding the immunogens, host cells, protein complexes or viral particles, or a combination thereof. The method may also include detecting the binding of antibodies to the disclosed immunogenic agents in the sample. The binding may be detected by any means known to those skilled in the art, including using a labeled secondary antibody that specifically binds to the antibody from the sample. Labels include radioactive labels, enzyme labels and fluorescent labels. In some embodiments, the method may further include separating HIV-1 binding antibodies in the subject.

[0118] The disclosed immunogenic agents can be used as components of a kit. Such a kit may also include other components, including packaging, instructions, and various other reagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents. The kit may optionally include an adjuvant.

[0119] Adjuvant is a vehicle for enhancing antigenicity. Adjuvant includes a mineral (alum, aluminum hydroxide or phosphate) suspension on which the antigen is adsorbed; or a water-in-oil emulsion, wherein the antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), sometimes containing inactivated mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (suppress antigen degradation and / or cause macrophage influx). Immunostimulatory oligonucleotides (such as those comprising CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent No. 6,194,388; U.S. Patent No. 6,207,646; U.S. Patent No. 6,214,806; U.S. Patent No. 6,218,371; U.S. Patent No. 6,239,116; U.S. Patent No. 6,339,068; U.S. Patent No. 6,406,705; and U.S. Patent No. 6,429,199). Adjuvants include biological molecules ("biological adjuvants"), such as co-stimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-a, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41BBL. Adjuvants can be used in combination with the disclosed immunogens.

[0120] IV. Definitions

[0121] As used in this document, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. All technical and scientific terms used herein are used unless defined otherwise.

[0122] The term "and / or" means any one, any combination of items, or all of the items associated with the term.

[0123] The compositions of the present invention may comprise, consist essentially of, or consist of the claimed ingredients. The words "comprise" (and any form of comprises, such as "comprises" and "comprises"), "have" (and any form of has, such as "have" and "has"), "include" (and any form of includes, such as "includes" and "include"), or "contain" (and any form of contains, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0124] The terms "treat" or "treatment" refer to the administration of a compound or agent to a subject suffering from or at risk of developing a disorder, with the intent to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, symptoms of the disorder, secondary disease states of the disorder, or a predisposition to the disorder.

[0125] The terms "prevent," "preventing," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of developing the disorder or condition.

[0126] The term "subject" refers to humans and non-human animals. Examples of non-human animals include all vertebrates, such as mammals, such as non-human mammals, non-human primates (especially higher primates), dogs, rodents (such as mice or rats), guinea pigs, cats and rabbits, and non-mammals, such as birds, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental non-human animal or an animal suitable as a disease model.

[0127] As disclosed herein, multiple value ranges are provided.It should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed, to one tenth of the lower limit unit, unless the context clearly stipulates otherwise.Each smaller range between any specified value or intermediate value in the specified range and any other specified value or intermediate value in the specified range is included in the present invention.The upper and lower limits of these smaller ranges can be independently included in the scope or excluded outside the scope, and wherein two limits are either, both include, and each range that is not included in the smaller range is also included in the present invention, provided that the limit specifically excluded in the specified range is not considered.In the case where the specified range includes one or two limits, the scope that does not include one or both of these included limits is also included in the present invention.

[0128] The term "about" generally refers to plus or minus 10% of the specified number. For example, "about 10%" can mean a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" may be obvious from the context, such as rounding, so, for example, "about 1" can also mean 0.5 to 1.4.

[0129] gp120 is an envelope protein from the human immunodeficiency virus (HIV). The mature gp120 wild-type polypeptide has approximately 500 amino acids in the primary sequence. gp120 is heavily N-glycosylated, resulting in an apparent molecular weight of 120 kD. The polypeptide consists of five conserved regions (C1-C5) and five highly variable regions (V1-V5). An exemplary sequence of a wild-type gp160 polypeptide is shown in , e.g., accession numbers AAB05604 and AAD12142, available as of June 29, 2010, which are incorporated herein by reference in their entirety. An exemplary sequence of a gp120 polypeptide from HIV-1 DU156 is shown at 53635, AAO50350 and AAT91997, which are available from September 27, 2010 and are incorporated herein by reference in their entirety. An exemplary sequence of a gp120 polypeptide from HIV-1 ZA012 is shown at See, e.g., Accession No. ACF75939, available as of September 27, 2010, which is incorporated herein by reference in its entirety.

[0130] "Glycosylation site" refers to an amino acid sequence on the surface of a polypeptide, such as a protein, that accommodates the attachment of a glycan. An N-linked glycosylation site is a triplet sequence of NXS / T, where N is asparagine, X is any residue except proline, and S / T means serine or threonine. A glycan is a polysaccharide or an oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or proteoglycan.

[0131] An "immunogenic polypeptide" refers to a protein or portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected with a pathogen or at risk of infection with a pathogen. Administration of an immunogenic polypeptide derived from a target pathogen induces an immune response. Administration of an immunogenic polypeptide can result in protective immunity against the target pathogen. In some examples, the immunogenic polypeptide is an antigen that has been resurfaced to focus immunogenicity to a target epitope. An "immunogenic gp120 polypeptide" is a gp120 molecule, a resurfaced gp120 molecule, or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal with or without HIV infection. Administration of an immunogenic gp120 polypeptide that induces an immune response can result in protective immunity against HIV.

[0132] "Immune response" refers to the response of cells of the immune system, such as B cells, T cells, or monocytes, to stimulation. In one embodiment, the response is specific for a particular antigen ("antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0133] "Isolated" means that an "isolated" biological component (e.g., a protein, such as a disclosed antigen or a nucleic acid encoding such an antigen) has been substantially separated or purified from other biological components in which the component naturally exists, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, and nucleic acids that have been "isolated" include proteins purified by standard purification methods. The term also encompasses proteins or peptides prepared by recombinant expression in a host cell, as well as chemically synthesized proteins, peptides, and nucleic acid molecules. Isolated (or purified) does not require absolute purity, and may include at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated proteins, peptides, or nucleic acid molecules.

[0134] "Encoding" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for synthesizing other polymers and macromolecules in a biological process, which have a determined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a determined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA produced by a gene produces a protein in a cell or other biological system, the gene encodes the protein. The coding strand of a gene or cDNA (whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and the non-coding strand (used as a transcription template) can both be referred to as encoding a protein or other product of the gene or cDNA. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns. In some instances, nucleic acids encode disclosed antigens. "Recombinant nucleic acid" refers to a nucleic acid having a nucleotide sequence that is not naturally connected together. This includes nucleic acid vectors that contain amplified or assembled nucleic acids and can be used to transform suitable host cells. Host cells containing recombinant nucleic acids are referred to as "recombinant host cells." The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide." The recombinant nucleic acid may also provide non-coding functions (eg, promoter, origin of replication, ribosome binding site, etc.).

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or necessary to those described herein may also be used in the practice or detection of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0136] V. Examples

[0137] Example 1

[0138] This example describes the materials and methods used in Examples 2-6 below.

[0139] Envelope protein

[0140] Env trimers were expressed as soluble native-like soluble gp140 trimers that included SOSIP substitutions: the “SOS” substitution (A501C gp120 、T605C gp41 ), "IP" (I559P gp41 ), residue 332 gp120 (T332N gp120 ), an enhanced gp120-gp41 cleavage site (REKR (SEQ ID NO: 17) to RRRRRR (SEQ ID NO: 18)), and residue 664 gp41 The stop codon after RC1 (Env numbering according to HX nomenclature). The newly engineered Env trimers RC1, RC1-4fill, RC1-Avitag, RC1-Spytag, RC1-Glycan KO, RC1-Glycan KO-Avitag, RC1-Glycan KO-GAIA ("GAIA" disclosed as SEQ ID NO: 16) and RC1-GAIA ("GAIA" disclosed as SEQ ID NO: 16), wtBG505 and previously reported BG505 variants 11MUTB, 10MUT, 7MUT, 5MUT were cloned into the pPPPI4 expression vector using synthetic gene fragments (Integrated DNA Technologies (IDT)). Glycan variants RC1Δ301, RC1Δ332 and 11MUTBΔ301 were generated by site-directed mutagenesis (QuikChange Lightning Multi-site Directed Mutagenesis Kit, catalog #210515, Agilent Technologies).

[0141] The untagged version of the Env protein was used in ELISA (see ELISA section) and immunization of wild-type mice (see Animal section). The Spytag version of RC1-4fill was coupled to virus-like particles (VLP) and used for immunization of rabbits and macaques (see Production and coupling of VLP and Animal section). The Avitag version of RC1 and RC1-Glycan KO was biotinylated and used as bait in FACS (see Flow cytometry and single B cell sorting section).

[0142] Soluble Env trimers were expressed in HEK293-6E cells (National Research Council of Canada) or Expi293 cells (Life Technologies) by transient transfection and purified from cell supernatants by 2G12 or NIH45-46 immunoaffinity chromatography and size exclusion chromatography (SEC) as previously described (Wang, H. et al. Elife 6 (2017)). Proteins were stored at 4°C in 20mM Tris pH 8.0 and 150mM sodium chloride (TBS buffer). The immunogen with SpyTag was buffered in 20mM sodium phosphate pH 7.5, 150mM sodium chloride.

[0143] VLP production and conjugation

[0144] To connect to VLP, the C-terminal SpyTag sequence (13 residues) was added to RC1-4fill to form an irreversible isopeptide bond with the SpyCatcher protein (Zakeri, B. et al. Proc Natl Acad Sci USA 109, E690-697 (2012)). The gene encoding the phage AP205 coat protein to which the SpyCatcher protein is connected was a gift from Dr. Mark Howarth of the University of Oxford. SpyCatcher-AP205 VLPs were purified as described (Brune, KD et al. SciRep 6, 19234 (2016)), incubated with a 3-fold molar excess of RC1-4fill Env trimers with SpyTag, and separated from the coupled VLPs by SEC on a Superdex 200 column equilibrated with 20 mM sodium phosphate pH 7.5, 150 mM NaCl. Coupling of Env trimers was verified by negative stain EM and / or SDS-PAGE, and immunogen concentration was estimated by comparison with known amounts of free immunogen run on the same SDS-PAGE gel.

[0145] animal

[0146] Mice carrying Ig V(D)J genes (GL HL 121 knock-in mice), which encode iGL IgH and IgL corresponding to human PGT121 and 10-1074 broadly neutralizing antibodies (Escolano, A. et al. Cell 166, 1445-1458e1412, (2016)). 6-8 week old C57BL6 male mice from Jackson Laboratory were used for immunization. All animal procedures were performed in accordance with the protocol approved by the IACUC of Rockefeller University. Male and female GL HL121 knock-in mice or male C57BL6 wild-type mice were evenly distributed in each group and immunized intraperitoneally with 10 μg of soluble SOSIP envelope trimer (1:1 in Ribi adjuvant (Sigma)).

[0147] Six-month-old New Zealand white rabbits (Covance) were immunized. Rabbits were subcutaneously immunized with approximately 22 μg of RC1-4fill SOSIP Env trimer (RC1-4fill VLP) coupled to VLP (in ISCOM-like saponin adjuvant) (see adjuvant synthesis section). Serum samples were collected from mice and rabbits at week 0 and week 2 after immunization.

[0148] Eight 2- to 4-year-old rhesus macaques (Macaca mulatta) of Indian genetic origin were housed and cared for in a biosafety level 2 NIH facility in accordance with the Guide for Care and Use of Laboratory Animals NIH Report No. 82-53 (Department of Health and Human Services, Bethesda, Maryland, 1985). All animal procedures and experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of NIAID, NIH.

[0149] Animals were immunized subcutaneously (sc) to the medial forelegs and hind legs (4 sites / animal) with about 200 μg of RC1-4fill SOSIPEnv trimer (RC1-4fill VLP) coupled to VLPs, with IscoMPLA as adjuvant. Blood was drawn regularly to monitor serum neutralizing activity and serum antibody binding was characterized by ELISA. Lymph node biopsies were obtained from unimmunized macaques and immunized macaques 3 weeks after immunization.

[0150] Adjuvant synthesis

[0151] ISCOM-like saponin adjuvants (K. -Bengtsson et al., in Methods in Molecular Medicine, Vaccine Adjuvants: Preparation Methods and Research Protocols, DO'Hagan, ed. (Humana Press, Totowa, NJ, 2000), Vol. 42, pp. 239-258). Briefly, 20 mg / ml solutions of cholesterol (Avanti Polar Lipids 700000) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Avanti Polar Lipids 850355) were prepared in 20% MEGA-10 (Sigma D6277) detergent. Quil-A saponin (InvivoGen vac-quil) was dissolved in Milli-Q water at a final concentration of 100 mg / ml. All components were mixed in a ratio of 1:1:5 (cholesterol: DPPC: Quil-A) and then diluted with 1xPBS to a final cholesterol concentration of 1 mg / ml. For ISCOM-MPLA saponin adjuvant, 5mg / ml MPLA (Avanti 699800) solution was prepared in 20% MEGA-10 and the components were mixed in a ratio of 2: 1: 1: 10 (cholesterol: DPPC: MPLA: Quil-A). The solution was equilibrated overnight at RT and then dialyzed against 1xPBS using a 10k MWCO membrane (ThermoFisher 66456). The adjuvant solution was then sterile filtered, concentrated using a 50k MWCO Centricon spin filter (Millipore Sigma UFC905024), and further purified by fast protein liquid chromatography (FPLC) using a Sephacryl S-500 HR size exclusion column (GE Life Sciences 28-9356-06). Final adjuvant concentration was determined by cholesterol quantification (Sigma MAK043).

[0152] ELISA

[0153] ELISAs were performed for the following: SOSIPEnv trimer 11MUTB, RC1, 11MUTBΔ301, RC1Δ301, RC1-GAIA ("GAIA" disclosed as SEQ ID NO: 16), RC1-glycan-knockout (RC1-glycan KO), RC1-glycan KO-GAIA ("GAIA" disclosed as SEQ ID NO: 16), RC1Δ332, BG505), 10MUT, 7MUT, 5MUT or V3 loop-consensus C-peptide (SEQ ID NO: 10: KGKGKGKGKGCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHC) by direct coating of high binding 96-well plates (Corning #9018) with 50 μl per well of 2 μg / ml protein solution in 1xPBS overnight at 4°C. The plates were washed 3 times with wash buffer (1xPBS containing 0.05% Tween 20 (Sigma-Aldrich)) and incubated for 1 hour (h) at room temperature (RT) in blocking buffer (1xPBS containing 2% milk powder). Monoclonal antibodies or serum samples were added to the blocking buffer immediately after blocking and incubated for 2 hours at RT. Serum samples were analyzed at an initial dilution of 1:100 or 1:30 and 7 additional 3-fold serial dilutions. Mouse and human monoclonal antibodies (IgG) or human Fab were evaluated at the concentrations specified in the results section. The plate was washed 3 times with wash buffer and then incubated with anti-mouse IgG (Jackson ImmunoResearch #115-035-071), anti-human IgG heavy chain (Jackson ImmunoResearch #109-035-098), or anti-human Ig heavy and light chains (Jackson ImmunoResearch #109-036-088) conjugated to horseradish peroxidase (HRP) diluted 1:5000 in wash buffer. The plate was developed by adding HRP substrate, ABTS single solution (Life Technologies #00-2024), and absorbance was measured at 405 nm using an ELISA plate reader (FluoStar Omega, BMG Labtech).

[0154] In other ELISAs, high binding 96-well plates were directly coated with 50 μl of 20 μg / ml Fab in 1xPBS overnight at 4°C. The plates were washed 3 times with wash buffer and incubated in blocking buffer for 1 hour at RT. Immediately after blocking, the plates were incubated in 50 μl of RC1 or RC1-glycan KO-GAIA ("GAIA" disclosed as SEQ ID NO: 16) solution (at 2 μg / ml in blocking buffer) for 1 hour at RT. The plates were washed 3 times with wash buffer and incubated with 50 μl of a chimeric form of the CD4 binding site bNAb 3BNC60 (human Fab and mouse Fc) (serially diluted 3-fold from 5 μg / ml in blocking buffer) for 1 hour at RT. The plates were washed 3 times with wash buffer and incubated with HRP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch #115-035-071) for 1 hour at RT. Plates were washed and developed as described above.

[0155] Flow cytometry and single B cell sorting

[0156] Single cell suspensions were obtained from draining lymph nodes and spleens of immunized mice, and mature B cells were isolated by negative selection using anti-CD43 magnetic beads (MACS) according to the manufacturer's instructions.

[0157] Frozen PBMCs or cells from lymph node biopsies obtained from unimmunized and immunized macaques were thawed and washed in RPMI medium 1640 (1×) (Gibco #11875-093). Mouse or macaque cells were incubated on ice for 30 minutes with 100 μl of FACS buffer (PBS 1×, containing 2% fetal bovine serum and 1 mM ethylenediaminetetraacetic acid (EDTA)) containing 1:500 dilution of mouse (BD Biosciences #553142) or human (BD Biosciences #564219) Fc blocker, respectively.

[0158] RC1 and RC1-Glycan KO (RC1-AviBio) were prepared by incubating 5 μg of Avitag-biotinylated RC1 (RC1-AviBio) or Avitag-biotinylated RC1-Glycan KO (RC1-Glycan KOAviBio) with fluorescent streptavidin (1:200 diluted in 1x PBS) on ice for 30 min. + / RC1 glycan KO-) tetramer.

[0159] RC1 was isolated using RC1-AviBio coupled to streptavidin BV711 (BD Biosciences, #563262) and RC1-GlycanKOAviBio coupled to streptavidin-PE (BD Biosciences, #554061) as baits. + / RC1-glycan KO-mouse B cells. Isolation of RC1 using three baits + / RC1-Glycan KO-Rhesus B cells: RC1-AviBio coupled to streptavidin-PE and streptavidin AF647, and RC1-Glycan KO AviBio coupled to streptavidin BV605 (BD Biosciences, #563260). Tetramers were mixed with the following human or mouse antibody mixtures to a final concentration of 5 μg / ml for both.

[0160] Mouse cells were stained with the following fluorescent antibodies against mouse cell surface markers: anti-CD4 APC-eFluor780 (Invitrogen, #47-0042-82), anti-CD8 APC-eFluor780 (Invitrogen, #47-0081-82), anti-F4 / 80 APC-eFluor780 (Invitrogen, #47-4801-82), anti-NK1.1 APC-eFluor780 (Invitrogen, #47-5941-82), anti-CD11b APC-eFluor780 (eBioscience #47-0112-82), anti-CD11c APC-eFluor780 (eBioscience #47-0114-82), anti-Gr-1 APC-eFluor780 (Invitrogen, #47-5931-82), anti-B220 APC (Biolegend, #103212), anti-GL7 FITC (BD Biosciences #553666) and anti-CD95 BV421 (BD Biosciences #562633) were diluted 1:200, and the live / dead marker Zombie NIR (Biolegend, #77184) was diluted 1:400 in FACS buffer. Macaque cells were stained with the following anti-human antibodies: anti-CD16 APC-eFluor780 (Invitrogen, #47-0168-41), anti-CD8a APC-eFluor780 (Invitrogen, #47-0086-42), anti-CD3 APC-eFluor780 (Invitrogen, #47-0037-41), anti-CD14 APC-eFluor780 (eBiosciences, #47-0149-41), anti-CD20 PeCy7 (BD, #335793), anti-CD38 FITC (StemCelltechnologies, #60131FI), anti-IgG BV421 (BD Biosciences, #562581), anti-IgMPerCP-Cy5.5 (BD Biosciences, #561285) at a dilution of 1:200, and the live / dead marker Zombie NIR was diluted 1:400 in FACS buffer. Mouse or macaque cells were incubated with the corresponding antibody mixture containing RC1 and RC1-glycan KO bait on ice for 30 minutes, washed with FACS buffer and resuspended in 1 ml FACS buffer. Before sorting or analysis, the cell suspension was filtered through a 40 μM cell strainer.

[0161] Zombie NIR was isolated from mouse cell homogenate using FACS Aria III (Becton Dickinson). - / CD4 - / CD8 - / F4 / 80 - / NK1.1 - / CD11b - / CD11c - / B220 + / GL7 + / CD95 + RC1 + / RC1-Glycan KO - Single Cell and Isolation of Zombie NIR from Macaque Cell Homogenates - / CD16 - / CD8a - / CD3 - / CD14 - / CD20 + / CD38 + / IgG + / - / Dual RC1 + / RC1-Glycan KO - Single cell.

[0162] Single cells were sorted into individual wells of a 96-well plate containing 5 μl of lysis buffer (TCL buffer (Qiagen #1031576) containing 1% 2-β-mercaptoethanol). The plates were immediately frozen on dry ice and stored at -80°C.

[0163] Antibody sequencing and cloning

[0164] Thaw the 96-well plate containing single-cell lysates on ice. Purify single-cell RNA in designated clean areas using magnetic beads (RNAClean XP, #A63987 Beckman Coulter) according to the manufacturer's instructions. In the final step of the purification protocol, RNA was eluted from the magnetic beads using 11 μl of a solution containing (14.5 ng / μl random primers (Invitrogen, #48190-011), 0.5% tergitol (NP-40 type, 70% in H2O, Sigma-Aldrich, #NP40S-100ML), 0.6 U / μl RNase inhibitor (Promega #N2615) in nuclease-free water (Qiagen) and incubated at 65°C for 3 minutes. Subsequently, reverse transcription ( cDNA was synthesized with 10 μl of nuclease-free water (Invitrogen, #18080-044, 10'000 U). The cDNA was stored at -80°C or used for antibody gene amplification by nested polymerase chain reaction (PCR). To amplify antibody genes from single B cells, 10 μl of nuclease-free water was added to the cDNA-containing solution.

[0165] Mouse and macaque antibody genes were amplified by nested PCR as previously described (von Boehmer, L et al. Nat Protoc 11, 1908-1923 (2016)). PCR protocol: (annealing (°C) / extension (seconds) / number of cycles): first PCR (IgG IgH and Igλ): 46 / 55 / 50; second PCR (IgG IgH and Igλ): 50 / 55 / 50. The amplified heavy and light chain cDNAs were cloned into expression vectors (fragment antigen-binding (Fab) vectors) containing complete mouse or human IgG antibody constant regions or human heavy chain constant region 1 using the sequence- and ligation-independent cloning (SLIC) method (Li, MZ & Elledge, SJ Nat Methods 4, 251-256 (2007)).

[0166] Antibody production and purification

[0167] Ig was purified from 200 μl of mouse or macaque serum using Ab Spin Trap Protein G Sepharose columns (GE Healthcare, #28-4083-47) according to the manufacturer's instructions. Ig was eluted into 4 fractions, 200 μl each. The fractions containing Ig were buffer exchanged with PBS by overnight dialysis at 4°C (dialysis cassette 20000 MWCO Thermo Scientific, #66005).

[0168] For structural studies, mouse IgG and macaque His6-tagged Fab ("His6" disclosed as SEQ ID NO: 19) were expressed in HEK293-6E or Expi293 cells by transient transfection and purified from cell supernatants using protein A or G (GE Healthcare) (for IgG) or Ni-NTA (GE Healthcare) or Ni Sepharose 6 Fast Flow (GE Healthcare) (for Fab) chromatography and SEC (as described in Scharf, L et al. Cell 162, 1379-1390 (2015)). Mouse Fab was purified by lysis with ficin (Sigma) at 1-5 mg ml using a protocol modified from Thermo Scientific. - 1 digested IgG. Fab was purified by protein G (GE Healthcare) and SEC chromatography followed by Mono Q 5 / 50 (GE Healthcare) ion exchange chromatography as described (Diskin, R. et al., Nat Struct Mol Biol 17, 608-613). As described above, the common iGL of PGT121 and 10-1074bNAb was expressed as a His6-tagged Fab ("His6" is disclosed as SEQ ID NO: 19).

[0169] In vitro neutralization assay

[0170] The TZM-b1 assay was performed as described (Montefiori, DC Curr Protoc Immunol Chapter 12, Unit 1211 (2005).) Briefly, neutralization activity was calculated as a function of the reduction in Tat-induced luciferase expression in the TZM-b1 reporter cell line after a single round of viral infection.

[0171] SPR

[0172] SPR experiments were performed using Biacore T200 (Biacore). To determine the affinity for PGT121 / 10-1074iGLFab, protein A was immobilized on a CM5 chip (Biacore) by primary amine chemistry (Biacore manual) and 200 nM 8ANC195 G52K5Anti-Env IgG was injected into the experimental flow cell as described (Scharf, L. et al. Cell 162, 1379-1390 (2015)). A reference flow cell was prepared by injecting 200nM mG053 IgG that does not bind HIV Env. 1μM human Fc was injected to block the remaining protein A sites. After capturing 10μM SOSIP protein (RC1, 11MUTB or 10MUT), a concentration series of PGT121 / 10-1074iGLFab was injected (4-fold dilution from the highest concentration of 160μM for 10MUT, 2-fold dilution from the highest concentration of 150μM for 11MUTB and RC1), and the binding reaction was allowed to reach equilibrium. The flow cell was regenerated as described (Scharf, L et al. Cell 162, 1379-1390 (2015)) with 10 mM glycine pH 2.0 and 1 M guanidine hydrochloride at a flow rate of 90 μl / min. eq K was obtained by nonlinear regression analysis of the plot of (equilibrium binding response) versus the logarithm of the injected protein concentration. D , and the data were fit to a 1:1 binding model as described (Vaughn, et al. Biochemistry 36, 9374-9380 (1997)).

[0173] To measure relative binding of antibodies isolated from mouse and monkey, SOSIP Env trimers were immobilized on a CM5 chip via primary amine chemistry and selected Fabs were injected at 200 nM. The flow cell was regenerated with 10 mM glycine pH 2.0.

[0174] Cryo-EM sample preparation

[0175] RC1 complexed with 10-1074 was prepared by incubating purified RC1 with 10-1074 Fab and CD4 binding site (CD4bs) Fab at a 1:3:3 molar ratio (gp140 monomer: 10-1074 Fab: CD4bs Fab) overnight at room temperature. RC1-Fab complexes were separated by SEC in TBS (20mM Tris pH 8.0, 100mM NaCl) using a Superdex-200 Increase 10 / 300 column (GE Healthcare). Complexes of RC1 with mouse or macaque Fab and 8ANC195 Fab were prepared by incubating purified RC1 with mouse or macaque Fab and 8ANC195 Fab at a 1:1.3:1.3 molar ratio (gp140 monomer: mouse or macaque Fab: 8ANC195 Fab) overnight at room temperature and used without SEC purification. The RC1-Fab complex was diluted to 0.75-1.4 mg / mL in TBS, added to glow-discharged 300 mesh Quantifoil R1.2 / 1.3 copper grids, and vitrified in liquid ethane using a Mark IV Vitrobot (FEI).

[0176] Cryo-EM data collection

[0177] The RC1-Fab complex was imaged on a Talos Arctica cryo-electron microscope, operated at 200 kV and equipped with a Falcon 3EC direct electron detector using EPU automated image acquisition software (Tan, et al. Microscopy (Oxf) 65, 43-56 (2016)). The RC1-10-1074 data were collected on two different days and merged at the time of processing. Each micrograph was collected at a magnification of 73,000, resulting in a pixel size of

[0178] Cryo-EM data processing

[0179] Movie micrographs were motion corrected in RELION-3 and dose weighted using MotionCor2, CTFs were estimated using Gctf, and particles were picked out from micrographs using Gaussian blob auto-picking (Zivanov, J. et al. Elife 7 (2018); Zheng, SQ et al. Nat Methods 14, 331-332 (2017); Zhang, K. Gctf. J Struct Biol 193, 1-12 (2016)). The extracted particles were imported into cryoSPARC v2 and classified into 2D class averages (Punjani, A., et al. Nat Methods 14, 290-296 (2017)). The selected particles were sorted into two ab initio models, and the selected models were used as references for homogenous refinement of those selected particles. Resolution was estimated using the Gold Standard Fourier shell correlation of an independently refined half-map (where FSC = 0.143), and the maps were automatically sharpened in cryoSPARC (Punjani, A., et al. Nat Methods 14, 290-296 (2017); Scheres, SH & Chen, S. Nat Methods 9, 853-854 (2012)). To resolve N-linked glycans, a series of maps were generated with an overall B factor range of -150 to - To improve the local features and map connectivity of PNGS (Terwilliger, TC, et al. Acta Crystallogr D Struct Biol 74, 545-559 (2018)).

[0180] Model building

[0181] The coordinates of each component of each complex were docked into the map using UCSF Chimera. For the RC1-10-1074 complex, BG505 (PDB 5T3Z), 10-1074 Fab (PDB 5T3Z) and 8ANC131 Fab (PDB 4RWY) were docked into the density (Goddard, TD, et al. J Structure Biol 157, 281-287 (2007)). For the complex of mouse or macaque Fab with RC1, BG505Env (PDB 5CEZ), PGT121 / 10-1074iGL Fab (PDB 4FQQ) and 8ANC195 Fab (PDB 5CJX) coordinates were docked into the density map. After replacing the sequences of Fab and RC1 in the complex, the model was built after iterative refinement in Coot and Phenix (Adams, PD et al. Acta Crystallogr D Biol Crystallogr 66, 213-221 (2010); Emsley, P., et al. Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010)). The coordinates of the glycans were added with Man9 and then adjusted to fit the map at σ = 5. Model validation was performed using MolProbity and Privateer (Chen, VB et al. Acta Crystallogr D Biol Crystallogr 66, 12-21 (2010); Agirre, J. et al. Nat Struct Mol Biol 22, 833-834 (2015)).

[0182] The CD4 binding site Fab in the RC1-10-1074 complex and the 8ANC195 Fab in complexes of RC1 with mouse and cynomolgus Fabs are not shown in the structure figures, and their coordinates are not included in the RC1-Fab complex structures deposited in EMDB and PDB.

[0183] Analysis software

[0184] Sequence analysis was performed using Geneious X and MacVector 15.5.3, and figures were created using R. Flow cytometry data were processed using FlowJo 10.5.0. Data analysis was performed using GraphPad Prism 7.

[0185] Quantitative and statistical analysis

[0186] Statistical information including n, mean, and statistical significance values ​​are presented in the text or figure legends. Statistical analysis was performed by unpaired T-test using GraphPad Prism 7. Data were considered statistically significant at *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.

[0187] Example 2

[0188] RC1 promotes antibody binding to the V3-glycan epitope

[0189] RC1 was designed using 11MUTB, a modified native-like soluble Env trimer derived from clade A / E BG505 Env (SOSIP.664), as a template. Compared to BG505, 11MUTB contains multiple substitutions in V1 and lacks potential N-linked glycosylation sites (PNGS) at positions N133 and N137 ( Figure 1a )(Steichen, JM et al. Immunity 45, 483-496 (2016); Sanders, RW et al. PLoS Pathog 9, e1003618 (2013)). It was hypothesized that additional removal of PNGS at position 156 (N156Q) would promote recognition of the V3-glycan patch by increasing the accessibility of the V1 portion interacting with the V3-glycan patch bNAb. Consistent with this idea, the loss of N156 PNGS enhanced the neutralization effect of PGT121 and 10-1074 (Figure 6a). In addition, we further hypothesized that removal of the N156 glycan (including the negatively charged terminal sialic acid) would generate a more electrostatically-neutral Env surface that could facilitate binding of the V3-glycan bNAbs PGT121 and the primary neutralizing antibody precursor of 10-1074 (iGL PGT121 / 10-1074).

[0190] RC1 was first characterized by evaluating its interaction with bNAbs by ELISA. As expected, V1-V2-specific bNAbs that interacted with the N156 glycan showed reduced binding to RC1 compared to BG505 (Figures 6a and 6b). In contrast, bNAbs targeting the V3-glycan epitope, the CD4 binding site, or the gp120-gp41 interface showed similar binding to RC1 and BG505 (Figure 6b). Therefore, RC1 retains the overall antigenic properties of BG505.

[0191] To further characterize RC1, the Fab of RC1 in complex with the antigen-binding fragment (Fab) of 10-1074 was resolved. Single-particle cryo-EM structures were obtained and compared with the structures of the same bNAbs bound to BG505 (Fig. 1b; Fig. 7; Table 3). The RC1 structure is similar to BG505, both showing a closed conformation of Env and containing three 10-1074 Fabs bound to three V3-glycan patch epitopes (Fig. 1b). Compared to BG505, the V1 loop in RC1 contains more ordered residues and shifts toward the CDRH3 of 10-1074, thereby increasing the interaction between RC1 and 10-1074 (Fig. 1b).

[0192] Despite the structural changes in V1 attributed to the loss of the N156 glycan (Fig. 1b), the common iGL precursors of PGT121 and 10-1074 bound to the cytoplasm with similar affinities (K D 50 μM) bound to RC1 and 11MUTB (Fig. 1c). Consistent with these observations, RC1 and 11MUTB elicited comparable V3-glycan epitope-specific serological responses in knock-in (KI) mice carrying the gene encoding iGL PGT121 / 10-1074 (Figs. 2a and 2b). In summary, RC1 exhibits structural changes compared to BG505, but these do not affect its affinity for the iGL PGT121 / 10-1074 precursor antibody.

[0193] Example 3

[0194] RC1 elicits V3-glycan plaque antibodies in wild-type mice

[0195] To determine whether RC1 could activate B cells carrying V3-glycan plaque-specific antibodies in wild-type mice, C57B1 / 6 mice were immunized with RC1 or 11MUTB. 11MUTB did not produce a measurable serological response (Figure 2c). In contrast, mice immunized with RC1 showed a reproducible anti-V3-glycan plaque response, as determined by ELISA comparing binding to RC1 and to a mutant RC1 lacking the two additional V3 PNGS at positions 301 and 332 (RC1-glycan KO) (Figures 2c, 2d, 2e, and 2f; Table 2). In addition, sera from mice immunized with RC1 cross-reacted with 11MUTB, but not with the more natural 10MUTEnv or BG505 ( Figure 8 ). The increased immunogenicity of the V3-glycan epitope of RC1 is a result of the specific removal of the N156 glycan from 11MUTB, as removal of the N301 glycan from 11MUTB (11MUTBΔ301) (see Table 2) failed to induce a detectable serological response in mice (Figure 2g). It was concluded that, unlike 11MUTB and 11MUTBΔ301, RC1 elicited a V3-glycan-specific serological response in wild-type mice.

[0196] To reduce the antibody response to off-target epitopes and further focus the response on the V3-glycan patch, the RC1 variant RC1-4fill was generated by adding PNGS to mask potential glycan off-target sites at gp120 positions 230, 241, 289 and 344 (Figure 9). The serological response elicited by RC1-4fill was more specific to the V3-glycan patch in wild-type mice than the serological response elicited by RC1, as determined by ELISA against RC1 and RC1-glycan KO (Figure 2h). It was concluded that RC1-4fill focused the antibody response to the V3-glycan patch epitope.

[0197] Example 4

[0198] Clonal expansion of V3-glycan plaque-specific B cells in wild-type mice

[0199] To further characterize the humoral responses elicited by RC1 and RC1-4fill in wild-type mice, antibody genes from single GC B cells that bound RC1 but not RC1-glycan KO were sequenced ( Fig.10 ). All mice immunized with RC1 and RC1-4fill analyzed showed expansion of GC B cell clones (Figure 2i). The expanded clones mainly expressed heavy chain V gene segments VH5-6, VH9-3 and VH2-9, and light chain segments VK3-4 and VK14-111 (Figure 2i; Tables 4, 5 and 6). The CDRH3 sequences in the expanded clones showed similarities with human V3-glycan plaque bNAbs, such as Tyr-rich or RxY motifs (Tables 4 and 6) and longer-than-average CDRH3, but none of them had insertions or deletions. The VH genes of the expanded clones had an average of 3.2 nucleotide mutations (Figure 2j; Table 4).

[0200] To determine the target sites of the antibodies produced by the expanded B cell clones, selected antibodies were cloned and produced and subjected to ELISA against RC1 and RC1 mutant proteins. A panel of different monoclonal antibodies (mAbs) showed specific binding to V3-glycan patches in the ELISA (Figure 2k). MUR ) or RC1-4fill(Ab276 MUR Further characterization of the Env binding properties of two mAbs isolated from mice immunized with ) showed that these antibodies bind to the V3-glycan epitope in a GDIR (SEQ ID NO: 15)- and N301-glycan-dependent manner (Figure 21; Table 2). Both antibodies bind to 11MUTB, but neither binds to BG505 or to the peptide that masks the crown of the V3 loop (Figure 21; Figure 11a). Ab275 MUR With a K of about 30 nM DBinds to RC1 (Figure 11b). Importantly, Ab275 MUR Retained binding to 11MUTB (K D The obtained mutation is critical for binding, as reverting to the iGL sequence results in loss of binding to RC1 (Figure 11d). As expected, Ab275 MUR and Ab276 MUR Neither showed detectable neutralizing activity against a panel of tier 1B and tier 2 HIV-1 isolates in the TZMb1 assay (data not shown). It was therefore concluded that RC1 and RC1-4fill expanded mouse B cell clones expressing antibodies targeting the V3-glycan patch.

[0201] Example 5

[0202] VLP-RC1-4fill elicits V3-glycan plaque antibodies in rabbits and rhesus monkeys

[0203] To enhance potential avidity effects and limit exposure of additional off-target epitopes at the base of the Env trimer, RC1-4fill was multimerized on virus-like particles (VLPs) using the Spytag-SpyCatcher system (Figures 3a and 3b). Rabbits and rhesus monkeys are considered better models than mice for HIV-1 vaccine research because their antibodies have longer CDRH3s than mice (an average of 11 residues in mice, 13 in rabbits, and 15 in rhesus monkeys and humans).

[0204] Immunization of 4 rabbits and 8 rhesus macaques with RC1-4fill VLPs elicited serological responses that were partially specific for V3-glycan plaques in all animals, as determined by ELISA against RC1 and RC1-glycan KO (Figures 3c and 3d). Sera from macaques primed with RC1-4fill VLPs showed sequentially decreasing binding to the more natural-like immunogens 11MUTB and 10MUT (Figure 3c and 3d). Fig.12 ). Thus, RC1-4fill VLPs elicited a robust serological response that localized to V3-glycan plaques in rabbits and rhesus monkeys.

[0205] To further characterize the RC1-4fill VLP-induced responses in macaques, draining lymph node GC B cells that bound RC1 but not RC1-glycan KO (RC1 + / RC1-glycan KO-). Although RC1 is absent in GC of unimmunized macaques + cells, RC1 was found in all GC B cells in the lymph nodes of the four macaques analyzed+ The average frequency of / RC1-glycan KO-GC B cells was 0.4% (Figures 3e and 3f).

[0206] Antibody clones from four immunized macaques showed that all displayed expanded B cell clones that used multiple VH genes with an average of 5.6 nucleotide somatic mutations (Figures 3g and 3h; Table 7). The most characterized human V3-glycan plaque bNAbs contained a lambda light chain. Analysis of lambda gene usage showed that macaque RC1 binding cells preferred to use genes VL132, which was 90.6% identical to VL2-8 in PGT125-128 and PGT130-131, and VL124, which was 93.8% identical to VL3-21 in PGT121-123 / 10-1074 (Figure 3i; Table 8). In addition, 86% of the lambda light chains had CDRL3, which included the DSS motif present in the iGLs of PGT121-123, 10-1074, and PGT124 (Figure 3j; Table 9). This motif mutated to DSR in the mature bNAb, and this substitution was essential for the neutralizing activity of PGT121. In conclusion, immunization of macaques with RC1-4fill VLPs expanded B cell clones with antibody sequences similar to the human V3-glycan plaque bNAb precursor.

[0207] Thirty-eight macaque GC antibodies were expressed with CDRL3s similar to those of the iGLV3-glycan plaque bNAbs (Table 10). The CDRL3s of 33 of these antibodies contained a DSS motif and a Q at position 89 (QxxDSS motif (SEQ ID NO: 20)), which is also found in the CDRL3s of PGT121-3, 10-1074, PGT124, and BG18 iGLs (Table 11). The other five antibodies contained a SYAG motif (SEQ ID NO: 21), which is present in the CDRL3s of PGT125-7, PGT128, PGT130, and PGT131 iGLs (Table 11). Thirty of the 33 antibodies containing the QxxDSS motif ("QxxDSS" disclosed as SEQ ID NO: 20) and two of the five antibodies containing the SYAG motif ("SYAG" disclosed as SEQ ID NO: 21) bound to the V3-glycan epitope as determined by ELISA using RC1 and RC1-glycan KO-GAIA ("GAIA" disclosed as SEQ ID NO: 16) (Figure 4a; Table 10). The CDRH3 lengths of these 38 V3-glycan antibodies ranged from 11 to 21 residues (average = 15.5 residues) (Figure 4b). The longer CDRH3s included a high content of Tyr and / or Phe residues, similar to the long CDRH3s of human V3-glycan bNAbs (Table 10). The VH and VL genes of these antibodies had an average of 4.9 and 3.3 nucleotide mutations, respectively (Figure 4c).

[0208] To further characterize antibody recognition of RC1, ELISA was performed for other mutants RC1-Glycan KO, RC1-GAIA ("GAIA" disclosed as SEQ ID NO: 16), RC1-Glycan KO-GAIA ("GAIA" disclosed as SEQ ID NO: 16), 11MUTBΔ301, RC1Δ301, RC1Δ332, 11MUTB and BG505 (Figures 4d and 4e; Table 2). The ELISA demonstrated four different binding patterns to RC1 between antibodies containing the QxxDSS motif (SEQ ID NO: 20) in CDRL3 (Figure 4d), and other patterns between antibodies containing the SYAG motif (SEQ ID NO: 21) (Figure 4e). Although all antibodies were glycan dependent (determined by lack of binding to RC1-glycan KO), they differed in their binding to 11MUTB or 10MUT, dependence on the GDIR motif (SEQ ID NO: 15), and dependence on the N301, N332, and N156 glycans (Figures 4a, 4d, and 4e). In the TZMb1 assay, none of the antibodies tested bound to BG505 or had neutralizing activity against a small panel of class 1B and class 2 HIV-1 isolates (Figures 4d and 4e; data not shown). It was concluded that immunization of macaques with RC1-4fill VLPs elicited V3-glycan plaque-specific antibodies that were similar to human bNAb precursors targeting this site.

[0209] Example 6

[0210] Cryo-EM structures of mouse and macaque antibodies in complex with RC1

[0211] To define the molecular mechanism of binding and compare the mode of V3-patch recognition, the structure of one mouse Fab and two macaque Fabs in complex with RC1 was determined using single-particle cryo-electron microscopy. All three antibodies bound to the V3-patch epitope with footprints that overlapped with the 10-1074 footprint, but at different angles of approach compared to 10-1074 (Figures 5a and 5b). Ab275 MUR ( resolution) and Ab874 NHP (derived from Ab876 NHP identical clones) bound similarly to each other, consistent with their 69% sequence identity, whereas Ab897 NHP (respectively with Ab275 MUR and Ab874 NHP related at 48% and 54% sequence identity) adopt different angles of approach (Fig. 5b).

[0212] All three Fabs in the RC1 complex contact the GDIR motif (SEQ ID NO: 15), but have different footprints compared to each other and 10-1074. 10-1074 uses CDRH3, CDRL1 and CDRL3 to contact the conserved GDIR motif (SEQ ID NO: 15) (Figures 1c and 5b), while Ab874 MUR and Ab275 NHP GDIR (SEQ ID NO: 15) contacts were made primarily using its CDRH2, and Ab897 NHP CDRL1 and CDRL3 were used (Figures 5b and 5c). In addition to contacting GDIR (SEQ ID NO: 15), Ab275 MUR and Ab874 NHP Ab275 also interacted with the N332 glycan (Figures 5a and 5b). However, unlike 10-1074, which interacted extensively with the N332 glycan through its CDRL1, FRWL3, CDRH2, and CDRH3, Ab275 MUR Minimal contact was achieved using only its CDRH2, and Ab874 NHP It is bound to the N332 glycan via its CDRH2 and FRWH3. NHP -No interaction with the N332 glycan was observed in the RC1 structure. MUR 、Ab876 NHP (with Ab874 NHP same clone) and Ab897 NHP Binding to RC1Δ301 was reduced (Figure 21), but none of the Fabs in the RC1 complex showed interaction with the N301 glycan, suggesting glycan heterogeneity that would mask this interaction and / or conformational changes in the V3-glycan patch that lack this glycan that would reduce binding. In conclusion, RC1 elicits antibodies targeting the V3-glycan patch with different binding modes in animals with polyclonal antibody repertoires, including primates.

[0213] HIV-1 bNAbs develop in infected humans through successive rounds of somatic mutation in response to a rapidly evolving pathogen. Vaccination with a range of relevant antigens recapitulates these events in genetically engineered mice carrying supraphysiological numbers of B lymphocytes expressing iGL precursors of bNAbs. An important goal of HIV-1 vaccine design is to design immunogens that elicit such responses in organisms with a polyclonal immune system, with the goal of reproducing these responses in humans.

[0214] HIV-1 vaccine immunogen design focuses on improving the affinity of candidate immunogens for specific iGL bNAb precursors, with the goal of recruiting a specific set of rare precursors to GCs. This approach generally fails to account for the increase in apparent affinity resulting from the interaction between multimeric antigens and multivalent antigen receptors on the surface of B cells. In addition, GC entry is primarily limited by competition. Therefore, the importance of affinity is relative, as demonstrated by the observation that B cells with low-affinity receptors are often found in GCs under physiological conditions.

[0215] The principle used to generate RC1 did not consider affinity. Instead, RC1 was designed to increase the number of bNAb progenitors that can compete with GC entry. This is achieved by making the antigen target site more available while promoting binding to the electrostatically neutral iGL precursor. In addition, RC1-4fill VLPs incorporate the idea that masking competition for off-target epitopes minimizes competition with GC entry.

[0216] RC1 differs from other HIV-1 vaccine candidates in that it induces B cells expressing antibodies against the targeted epitope to be clonally expanded in the GC of animals with a complete polyclonal B cell repertoire. In macaques, antibodies expressed by these B cells showed sequence and structural similarity to the iGL precursors of bNAbs targeting the V3-glycan patch. Therefore, RC1-4fillVLPs are a suitable candidate immunogen for sequential vaccination strategies aimed at eliciting V3-glycan bNAbs.

[0217] Example 7

[0218] RC1-3fill VLP and NP performed similarly to RC1 VLP and NP

[0219] The size exclusion chromatography (SEC) traces of RC1, RC1-3fill and RC1-4fill immunogens (Figure 13a) show that the fraction of RC1 and RC1-3fill immunogens eluted in the void volume is smaller than that of RC1-4fill, demonstrating that RC1 and RC1-3fill are more stable and less prone to aggregation than RC1-4fill. Representative yields of 1 L expression of each immunogen in HEK 293T 6E cells (Figure 13b) show that the expression level of RC1-3fill is higher than that of RC1-4fill and similar to that of RC1.

[0220] Figure 13c shows representative SEC traces of the purification of AP205-RC1-VLP (dark gray) and AP205-RC1-3fill-VLP (black). Figure 13d shows electron micrographs of AP205-RC1-VLP (left) and AP205-RC1-3fill-VLP (right), showing that AP205-RC1-3fill-VLP looks similar to AP205-RC1-VLP and has a similar number of coupled trimers per particle. The micrographs also show that the purification strategy is sufficient and that free trimers are not present in either sample. Representative SEC traces of the purification of mi3-RC1-NP (dark gray) and mi3-RC1-3fill-NP (black) (Figure 13e) show that RC1 and RC1-3fill can be coupled to mi3 NP. Electron micrographs of mi3-RC1-NP (left) and mi3-RC1-3fill-NP (right) ( FIG. 13 f ) show that mi3-RC1-3fill-NP looks similar to mi3-RC1-NP and has a similar number of coupled trimers per particle. The micrographs also show that the purification strategy is adequate and that free trimers are not present in either sample. Initial purification of AP205-RC1-VLP ( FIG. 13 g ) and mi3-RC11-NP ( FIG. 13 h ) and SEC spectra of reinjected samples at 28 days (AP205) and 11 days (mi3) show that the coupled particles are stable over time and that no uncoupled RC1 or degradation products were observed after 28 or 11 days of storage.

[0221] Sera from six WT mice immunized with mi3-RC1-NP (Figure 13i) or mi3-RC1-3fill-NP (Figure 13j) were tested for binding to RC1 (black) and RC1 glycan KO (grey). Sera from all six mice immunized with Mi3-RC1 or Mi3-RC1-3fill bound to RC1 in the ELISA, and binding to RC1 glycan KO was reduced, indicating a serum response specific for V3 / N332 glycan plaques. Monoclonal antibodies 10-1074 and 3BNC117 were included as positive and negative controls. ELISA data are shown as the area under the ELISA curve (AUC).

[0222] Table 3: Cryo-EM data collection and processing statistics

[0223]

[0224]

[0225] Table 4: Antibody sequences generated from mice immunized with RC1 and RC1-4fill

[0226]

[0227]

[0228]

[0229]

[0230] Table 5: Antibody sequences generated from mice immunized with RC1 and RC1-4fill

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238] Table 7: Antibody sequences generated from macaques immunized with RC1-4fill VLP

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] Table 7 (continued): Antibody sequences generated from macaques immunized with RC1-4fill VLP

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] Table 8: Use of lambda genes

[0257]

[0258] Table 9: Antibody sequences containing the DSS motif produced by macaques immunized with RC1-4fill VLP

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276] Table 11: bNAbs containing the QxxDSS motif ("QxxDSS" disclosed as SEQ ID NO: 20)

[0277]

[0278] Table 12: PCR Primers

[0279]

[0280]

[0281]

Claims

1. An isolated anti-HIV antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region (CDR) 3 (CDRH3) selected from the group consisting of SEQ ID NOs: 22-49 and 110-283 and a light chain CDR3 (CDRL3) selected from the group consisting of SEQ ID NOs: 50-95 and 284-406.

2. The isolated anti-HIV antibody or antigen-binding fragment thereof of claim 1, which binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 11 and 13.

3. The isolated anti-HIV antibody or antigen-binding fragment thereof according to claim 2, comprising any one of the following amino acid sequences 1) to 57): 1) SEQ ID NO: 96 and 65 (antibody 271); 2) SEQ ID NO: 97 and 109 (antibody (340); 3) SEQ ID NO: 98 and 84 (antibody 341); 4) SEQ ID NO: 99 and 78 (antibody 343); 5) SEQ ID NO: 100 and 95 (antibody 344); 6) SEQ ID NO: 101 and 78 (antibody 346); 7) SEQ ID NO: 102 and 70 (antibody 347); 8) SEQ ID NO: 103 and 50 (antibody 351); 9) SEQ ID NO: 104 and 61 (antibody 352); 10) SEQ ID NO: 105 and 70 (antibody 274); 11) SEQ ID NO: 105 and 61 (antibody 275); 12) SEQ ID NO: 40 and 65 (antibody 276); 13) SEQ ID NO: 40 and 67 (antibody 278); 14) SEQ ID NO: 46 and 65 (antibody 280); 15) SEQ ID NO: 106 and 70 (antibody 294); 16) SEQ ID NO: 107 and 93 (antibody 348); 17) SEQ ID NO: 108 and 94 (antibody 349) 18) SEQ ID NO: 134 and 294 (antibody 874); 19) SEQ ID NO: 133 and 294 (antibody 876); 20) SEQ ID NO: 182 and 285 (antibody 890); 21) SEQ ID NO: 145 and 291 (antibody 893); 22) SEQ ID NO: 172 and 433 (antibody 897); 23) SEQ ID NO: 195 and 285 (antibody 901); 24) SEQ ID NO: 407 and 314 (antibody 933); 25) SEQ ID NO: 408 and 284 (antibody 934); 26) SEQ ID NO: 409 and 362 (antibody 935); 27) SEQ ID NO: 410 and 394 (antibody 936); 28) SEQ ID NO: 411 and 311 (antibody 937); 29) SEQ ID NO: 412 and 348 (antibody 938); 30) SEQ ID NO: 413 and 395 (antibody 986); 31) SEQ ID NO: 414 and 400 (antibody 987); 32) SEQ ID NO: 415 and 402 (antibody 988); 33) SEQ ID NO: 416 and 286 (antibody 990); 34) SEQ ID NO: 417 and 284 (antibody 992); 35) SEQ ID NO: 418 and 284 (antibody 992a); 36) SEQ ID NO: 419 and 318 (antibody 996); 37) SEQ ID NO: 420 and 286 (antibody 997); 38) SEQ ID NO: 421 and 315 (antibody 998); 39) SEQ ID NO: 422 and 285 (antibody 1000); 40) SEQ ID NO: 423 and 373 (antibody 1002); 41) SEQ ID NO: 424 and 315 (antibody 1003); 42) SEQ ID NO: 425 and 382 (antibody 1004); 43) SEQ ID NO: 426 and 311 (antibody 1005); 44) SEQ ID NO: 427 and 305 (antibody 1013); 45) SEQ ID NO: 214 and 313 (antibody 1053); 46) SEQ ID NO: 219 and 284 (antibody 1054); 47) SEQ ID NO: 276 and 342 (antibody 1061); 48) SEQ ID NO: 252 and 322 (antibody 1062); 49) SEQ ID NO: 247 and 347 (antibody 1063); 50) SEQ ID NO: 257 and 355 (antibody 1064); 51) SEQ ID NO: 277 and 363 (antibody 1068); 52) SEQ ID NO: 428 and 383 (antibody 1169); 53) SEQ ID NO: 429 and 383 (antibody 1169a); 54) SEQ ID NO: 430 and 392 (antibody 1170); 55) SEQ ID NO: 272 and 352 (antibody 1177); 56) SEQ ID NO: 431 and 361 (antibody 1178); or 57) SEQ ID NO: 432 and 383 (antibody 1180).

4. A pharmaceutical composition comprising the isolated anti-HIV antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

5. A pharmaceutical composition for use in a method for preventing or treating HIV infection or HIV-related diseases, wherein the method comprises the following steps: Identify patients in need of such prevention or treatment; and A first therapeutic agent comprising a therapeutically effective amount of at least one isolated anti-HIV antibody or antigen-binding fragment thereof according to any one of claims 1-3 is administered to the patient.

6. The pharmaceutical composition of claim 5, wherein the method further comprises administering a second therapeutic agent.

7. A kit comprising a pharmaceutically effective amount of at least one isolated anti-HIV antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in a pharmaceutically acceptable dosage unit.

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