Immunogens and methods for inducing broadly neutralizing antibodies targeting the HIV envelope apex epitope

Abstract

The present invention provides compositions and methods for inducing broadly neutralizing antibodies (bnAbs) against HIV, specifically targeting the Apex epitope of the HIV envelope protein. The invention describes a novel immunogen, ApexGT6, an engineered HIV envelope trimer designed to bind precursors of Apex bnAbs. Methods of administering ApexGT6 as an adjuvanted protein or mRNA-lipid nanoparticle (LNP) to consistently induce Apex bnAb-related precursor B cells with long heavy chain complementarity determining region 3 (HCDR3) loops in non-human primates are disclosed. The invention further provides methods for evaluating the induced immune response, including assessing antibody affinity maturation and structural characterization of elicited antibodies. This technology represents a significant advancement in HIV vaccine development and may have broader applications in inducing antibodies with very long HCDR3s against other pathogens.

Description

PATENT Docket No. Y7969-99073 IMMUNOGENS AND METHODS FOR INDUCING BROADLY NEUTRALIZING ANTIBODIES TARGETING THE HIV ENVELOPE APEX EPITOPE RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims priority to US provisional patent application Serial No.63 / 726,783 filed December 2, 2024.

[0002] The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said.xml copy, created on December 02, 2025, is named Y7969-99073, and is 477,833 bytes in size.FEDERAL FUNDING LEGEND

[0004] This invention was made with government support under Grant Nos. UM1 AI100663, UM1 AI144462, R01 AI113867, and P51OD011132 awarded by the National Institute of Allergy and Infectious Diseases. The government has certain rights in the invention.FIELD OF THE INVENTION

[0005] The present invention relates generally to the field of vaccine development. More specifically, the invention relates to immunogens and methods for inducing broadly neutralizing antibodies against HIV, particularly antibodies targeting the apex epitope of the HIV envelope protein.1DM2\301045646.1PATENT Docket No. Y7969-99073 BACKGROUND OF THE INVENTION

[0006] Human Immunodeficiency Virus (HIV) continues to be a major global health concern, with millions affected worldwide. Despite decades of research and multiple vaccine strategies, an effective prophylactic vaccine remains elusive. One primary challenge in developing an HIV vaccine is the virus's remarkable ability to evade immune responses through its high mutation rate and dense glycan shield that protects conserved epitopes on the viral envelope protein. Traditional vaccine approaches have failed to elicit antibodies capable of neutralizing diverse viral strains, necessitating novel strategies for vaccine development.

[0007] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0008] Broadly neutralizing antibodies (bnAbs) have emerged as a promising target for HIV vaccine development. These antibodies can neutralize a wide range of HIV strains by targeting conserved regions of the viral envelope protein. Several classes of bnAbs have been identified, including those targeting the CD4 binding site, V3-glycan patch, MPER region, and the trimer apex. Among these, Apex bnAbs are particularly attractive due to their potency and relatively low levels of somatic hypermutation required for breadth. While CD4 binding site bnAbs typically require 30-40% somatic hypermutation to achieve breadth, Apex bnAbs can achieve similar breadth with only 10-20% mutation. However, these antibodies face a different challenge: they require exceptionally long heavy chain complementarity determining region 3 (HCDR3) loops to penetrate the glycan shield and access conserved protein residues.

[0009] Apex bnAbs are characterized by their exceptionally long heavy chain complementarity determining region 3 (HCDR3) loops, which allow them to penetrate the glycan shield and access conserved protein residues. However, B cells capable of producing such antibodies are rare in the naive B cell repertoire, making them challenging to induce through traditional vaccination approaches.

[0010] Recent advances in immunogen design have led to the concept of germline-targeting vaccines, which aim to activate and expand rare B cell precursors with the potential to develop2DM2\301045646.1PATENT Docket No. Y7969-99073 into bnAbs. This approach has shown promise in animal models and early-phase clinical trials for other HIV epitopes, such as the CD4 binding site.

[0011] The present invention builds upon these advances by providing a novel immunogen, ApexGT6, specifically designed to target and activate B cell precursors with the potential to develop into Apex bnAbs. This invention addresses the critical need for an immunogen capable of consistently inducing long HCDR3 antibody responses in outbred populations, a key step towards developing an effective HIV vaccine.

[0012] Furthermore, the methods described herein for delivering the immunogen as either an adjuvanted protein or mRNA-LNP provide flexibility in vaccination strategies. The use of mRNA-LNP technology, in particular, offers potential advantages in terms of manufacturing speed and cost-effectiveness, which could accelerate clinical development.

[0013] By demonstrating the successful induction of Apex bnAb-related precursors in nonhuman primates, this invention represents a significant advancement in HIV vaccine research. Moreover, the principles and methods described may have broader applications in developing vaccines against other challenging pathogens where long HCDR3 antibodies play a crucial role in neutralization.

[0014] The present invention provides engineered immunogens for inducing broadly neutralizing antibodies against HIV. In one aspect, the invention features a modified HIV envelope (Env) protein comprising one or more modifications, including mutations in the V1 / V2 region, mutations that enhance binding of the Env protein to precursors of broadly neutralizing antibodies (bnAbs) targeting the apex epitope, mutations that facilitate proper protein folding, mutations that enhance trimer stability, addition or removal of glycosylation sites, and combinations thereof. The modified Env protein induces B cells expressing antibodies that bind to the apex epitope of HIV Env.

[0015] In certain embodiments, the modified Env protein is provided in the form of a trimer, which may be either soluble or membrane-bound. The invention encompasses specific mutations in the V1 / V2 region, including P76S, T128G, and N195S, as numbered according to HXB2. In some embodiments, the modified Env protein may also comprise glycosylation sites at positions N241 and N289. In some embodiments, the modified Env protein is ApexGT6, specifically comprising the ApexGT6 congly sequence or variants thereof having at least 90% sequence identity.3DM2\301045646.1PATENT Docket No. Y7969-99073

[0016] In certain embodiments, the modified Env protein is provided in either soluble or membrane-bound trimer form. The invention provides both ApexGT6 congly for protein-based delivery and ApexGT6 L14 gpl 51 for mRNA-based delivery, as well as variants thereof having at least 90% sequence identity.

[0017] The invention further provides compositions comprising the engineered immunogen and a lipid nanoparticle, as well as pharmaceutical compositions comprising the immunogen and a pharmaceutically acceptable carrier. In some embodiments, the modified Env protein is encoded by an mRNA.

[0018] Methods of using the engineered immunogen are also provided, including methods of inducing an immune response against HIV by administering the immunogen to a subject. The immunogen may be administered as an adjuvanted protein or as an mRNA-LNP, and the methods may include administering one or more booster immunizations. In some embodiments, the induced immune response may comprise B cells expressing antibodies with HCDR3 lengths of 24 amino acids or greater, antibodies that utilize the IGHD3-15 gene segment, or antibodies containing a DDY motif.

[0019] The invention additionally encompasses isolated antibodies or antigen-binding fragments thereof that bind to the apex epitope of HIV Env, characterized by a heavy chain CDR3 of 24 amino acids or greater in length. In some embodiments, these antibodies may utilize the IGHD3-15 gene segment and bind to ApexGT6 with a KD of 10 nM or less.

[0020] Methods of identifying subjects with precursors to apex bnAbs are also provided, comprising obtaining a biological sample from a subject, contacting the sample with the engineered immunogen, and detecting binding of B cells to the immunogen. The invention further includes kits comprising the immunogen and instructions for administration.

[0021] The methods described herein provide unprecedented flexibility in vaccination strategies through dual delivery platforms: adjuvanted protein and mRNA-LNP formulations. The mRNA-LNP technology offers particular advantages in manufacturing speed and costeffectiveness, potentially accelerating clinical development. These delivery systems can be employed individually or in combination, enabling optimization of immune responses through prime-boost strategies. The invention further provides comprehensive methods for monitoring immune responses and identifying subjects likely to benefit from vaccination.4DM2\301045646.1PATENT Docket No. Y7969-99073

[0022] This invention represents a significant advancement in HIV vaccine research through successful demonstration of Apex bnAb-related precursor induction in non-human primates. The principles and methods described extend beyond HIV vaccination, offering potential applications in developing vaccines against other challenging pathogens where long HCDR3 antibodies play crucial roles in neutralization. The therapeutic and diagnostic applications of the elicited antibodies further enhance the invention's impact on HIV treatment and monitoring. The comprehensive nature of this technology platform, encompassing immunogen design, delivery strategies, and response monitoring, provides a template for addressing similar challenges in other disease contexts.

[0023] By demonstrating successful induction of Apex bnAb-related precursors in non-human primates, this invention represents a significant advancement in HIV vaccine research. Moreover, the principles and methods described may have broader applications in developing vaccines against other challenging pathogens where long HCDR3 antibodies play a crucial role in neutralization. The therapeutic and diagnostic applications of the elicited antibodies further enhance the invention's impact on HIV treatment and monitoring. The comprehensive nature of this technology platform, encompassing immunogen design, delivery strategies, and response monitoring, provides a template for addressing similar challenges in other disease contexts.

[0024] The invention also encompasses antigen-binding fragments conjugated to a chemically functional moiety. Typically, the moiety is a label capable of producing a detectable signal. These conjugated antigen-binding fragments are useful, for example, in detection systems such as quantitation of DCs in various tissues, in various diseases, after stem cell transplantation, and after immunoablative therapy like chemotherapy and radiation, and imaging of DCs for instance in following chemotherapy or autoimmune therapy. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, substrate cofactors and inhibitors and magnetic particles. For examples of patents teaching the use of such labels, see, for instance U. S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. The moieties can be covalently linked, recombinantly linked, or conjugated (covalently or non-covalently) through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex.

[0025] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such5DM2\301045646.1PATENT Docket No. Y7969-99073 that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U. S. C. §112(a)) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0026] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U. S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U. S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0027] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The patent or application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0030] FIG. 1. (SEQ ID NOs: 447-457) Structure-guided directed evolution produced improved Apex bnAb germline-targeting priming immunogens. FIG. 1A displays somatic hypermutation of bnAbs versus their HCDR3 length. FIG. IB shows the immunogen design pathway from ApexGT trimers to ApexGT6 congly gpl40 and ApexGT6 L14 gpl51. FIG. 1C6DM2\301045646.1PATENT Docket No. Y7969-99073 illustrates SPR KDs for MD39 and ApexGT trimer binding to PCT64 and PG9 variants. FIG. ID shows alignment of RM IGHD3-41 alleles and human IGHD3-3. FIG. IE demonstrates the frequency of PCT64-like HC sequences in human donors and RMs. FIG. IF displays alignment of RM IGHD3-15 and HCDR3 sequences. FIG. 1G shows PCT64 and RHA1 HCDR3s buried within trimers. FIG. 1H illustrates frequency of Apex bnAb-like HC sequences in humans and RMs.

[0031] FIG. 2. ApexGT6 adjuvanted-protein immunizations elicited strong Apex epitopespecific responses. FIG. 2A shows the experimental schematic. FIG. 2B displays ELISA quantification of epitope-specific binding serum IgG titers. FIGs. 2C-D show polyclonal plasma IgG binding analysis by nsEMPEM. FIGs. 2E-H demonstrate flow cytometry analysis of B cell responses.

[0032] FIG. 3. (SEQ ID NOs: 143-145,147,149,150,153- 155,158,211-217,219-228,454,458-460) Characterization of Apex bnAb-like precursor responses induced by ApexGT6 adjuvanted-protein immunizations. FIG. 3A shows HCDR3 length distribution. FIG. 3B displays IGHD3-15 frequency comparison. FIGs. 3C-D demonstrate percentages of Apex bnAb-like precursor BCRs. FIG. 3E shows alignment of IGHD3-15 and representative HCDR3 sequences.

[0033] FIG. 4. (SEQ ID Nos: 169-179,181,182,188-193,195-198,202,205-209,454,461-511) Analysis of membrane-bound ApexGT6 mRNA-LNP induced responses. FIGs. 4A-D show ELISA and flow cytometry analyses. FIGs. 4E-H display BCR sequence analyses. FIGs. 4I-K demonstrate comparative analyses between protein and mRNA-LNP immunization platforms.

[0034] FIG. 5. Characterization of antibody maturation and affinity development. FIGs. 5A-B show somatic hypermutation analysis. FIGs. 5C-E display SPR affinity measurements. FIGs.5F-G show V2b loop and strand C feature analyses. FIGs. 5H-J demonstrate binding to native-like Apex epitopes.

[0035] FIG. 6. Structural analysis of ApexGT6-induced antibodies. FIGs. 6A-G show cryo-EM structures and analyses of RM038 antibody binding to ApexGT6.

[0036] FIG. 7. Analysis of L- variant CHOI -04 bnAbs. FIGs. 7A-G show structural analyses. FIG. 7H displays precursor frequencies. FIG. 71 shows neutralization data. FIG. 7J demonstrates binding affinity measurements.

[0037] FIG. 8. Additional characterization of ApexGT immunogens. FIGs. 8A-H show binding properties, stability evaluation, and precursor definition analyses.7DM2\301045646.1PATENT Docket No. Y7969-99073

[0038] FIG. 9. Detailed analysis of epitope-specific responses elicited by ApexGT6 adjuvanted soluble protein. FIG. 9A shows ELISA quantification of serum IgG titers. FIG. 9B displays composite figures from nsEMPEM analysis. FIGs. 9C-D demonstrate the gating strategies used to identify epitope-specific BGC and Bmem cells, respectively.

[0039] FIG. 10. Extended analysis of Apex bnAb-like precursor responses from protein immunization. FIG. 10A shows the schematic for ApexGT6++B cell sorting and lOx GEM sequencing. FIG. 10B displays HCDR3 length distribution of BCRs in lymph nodes for each RM.FIG. 10C demonstrates frequency comparison of RM IGHD3-15 among GC BCRs.

[0040] FIG. 11. Comprehensive analysis of mRNA-LNP immunization responses. FIGs. 11 A-D show ELISA quantification and comparative analyses between immunization platforms. FIG.11E-F display HCDR3 length distributions. FIGs. 11G-H demonstrate correlation between HCDR3 length and neutralization properties.

[0041] FIG. 12. Detailed characterization of antibody maturation. FIG. 12A shows VH and VK / X somatic hypermutation patterns. FIG. 12B displays SPR affinity measurements. FIGs. 12C-D show V2b loop and strand C feature analyses. FIGs. 12E-F demonstrate binding characteristics to ApexGT6 variants.

[0042] FIG. 13. Additional structural analyses of RM038 antibody. FIGs. 13A-D show detailed views of DDY motif interactions. FIGs. 13E-G display structural comparisons with PG9 and analysis of hydrophobic pocket interactions.

[0043] FIG. 14. (SEQ ID NOs: 512-517) Supplementary structural data for RM018 antibody. FIG. 14A shows structural comparison withRM038. FIGs. 14B-C display DDY motif interactions. FIG. 14D shows neutralization assay results. FIG. 14E demonstrates sequence alignments of tested antibodies.

[0044] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention provides novel immunogens, compositions, and methods for inducing broadly neutralizing antibodies (bnAbs) that specifically target the apex epitope of the HIV envelope glycoprotein (Env). The immunogens disclosed herein, particularly ApexGT68DM2\301045646.1PATENT Docket No. Y7969-99073 variants, have been designed to engage rare B cell precursors capable of developing into bnAbs with long heavy chain complementarity determining region 3 (HCDR3) loops. Such bnAbs, including those based on apex-targeting prototypes like PCT64 and PG9, allow the neutralization of a wide array of HIV- 1 strains.

[0046] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0047] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0048] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as values between 10 and 15. For example, it is also understood9DM2\301045646.1PATENT Docket No. Y7969-99073 that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In this application, if a data point range is disclosed, it is understood that each unit from the lowest data point to the highest stated datapoint, including the first (lowest) and last (highest) data point is disclosed. For example, if a data point range 1-20 is disclosed, it is understood that data points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and each unit between any two particular units in the range are also disclosed. It is also understood that whenever a series of values are disclosed, that any range falling between any two of the recited values is also understood to be included.

[0049] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0050] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes embodiments where said event or circumstance occurs and embodiments where it does not.

[0051] The immunogens described herein serve multiple purposes in addressing HIV infection. As vaccine components, they can elicit broadly neutralizing antibodies targeting viral epitopes. In diagnostic applications, they enable detection and monitoring of immune responses. The antibodies elicited by these immunogens can be used therapeutically to treat HIV infection and diagnostically to detect viral presence. The therapeutic applications encompass both preventative and treatment modalities. For prevention, the antibodies can be administered before potential exposure, providing temporary protection. In treatment settings, the antibodies can be used alone or in combination with other antiretroviral therapies. Therapeutic administration typically employs doses ranging from 1-50 mg / kg, with optimal responses often seen in the 2-20 mg / kg range. The dosing interval can be adjusted based on pharmacokinetic studies and clinical response, typically ranging from 2-8 weeks.

[0052] As used herein, the term “immunogen” or “antigen” refers to a substance, typically a protein, capable of inducing an immune response in a subject. In the context of this invention, this specifically includes modified HIV envelope (Env) proteins engineered to induce broadly neutralizing antibodies targeting the apex epitope. These terms are used interchangeably when referring to proteins that are immunologically active and capable of evoking humoral and / or cellular immune responses when administered to a subject, either directly or through nucleotide sequences encoding the protein.10DM2\301045646.1PATENT Docket No. Y7969-99073

[0053] “Broadly neutralizing antibody” or “bnAb” refers to an antibody capable of neutralizing multiple strains or variants of HTV by binding to conserved epitopes on the viral envelope protein. In the context of this invention, apex bnAbs specifically target the V1 / V2 region at the apex of the HIV envelope trimer.

[0054] An antibody or Ab is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen binding portion (e.g., antigen-binding fragment) thereof of an intact antibody that retains the ability to specifically bind to a given antigen or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site, for example without limitation, Fab; Fab'; F(ab')2; an Fd fragment; an Fv fragment; a single domain antibody (dAb) fragment; an isolated complementarity determining region (CDR); single chain (scFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.

[0055] Hudson, 2005, Nature Biotechnology 23(9): 1 126-1 136). An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2. The heavy chain (HC) constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0056] An isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds a target may, however, have cross-reactivity to other antigens, such as the same target molecule from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.11DM2\301045646.1PATENT Docket No. Y7969-99073

[0057] A variable region of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FRs) connected by three complementarity determining regions (CDR1, CDR2, CDR3) also known as hypervariable regions, contribute to the formation of the antigen binding site of antibodies. If variants of a subject variable region are desired, particularly with substitution in amino acid residues outside of a CDR region (i.e., in the framework region), appropriate amino acid substitution, preferably, conservative amino acid substitution, can be identified by comparing the subject variable region to the variable regions of other antibodies which contain CDR1 and CDR2 sequences in the same canonincal class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4): 901 -917, 1987). When choosing FR to flank subject CDRs, e.g., when humanizing or optimizing an antibody, FRs from antibodies which contain CDR1 and CDR2 sequences in the same canonical class are preferred.

[0058] A CDR of a variable domain are amino acid residues within the variable region that are identified in accordance with the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, and / or conformational definitions or any method of CDR determination well known in the art. Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D. C. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, e g., Chothia et al., 1989, Nature 342:877-883. Other approaches to CDR identification include the AbM definition, which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the contact definition of CDRs based on observed antigen contacts, set forth in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. In another approach, referred to herein as the conformational definition of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1 156-1 166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.12DM2\301045646.1PATENT Docket No. Y7969-99073 As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and / or conformational definitions.

[0059] The terms IgG Fc region, Fc region, Fc domain and Fc, as interchangeably used herein refer to the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region consists of the C-terminal half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the binding sites for complement and Fc receptors, including the FcRn receptor (see below). The Fc fragment contains the entire second constant domain CH2 (residues 231 -340 of human IgGl, according to the Kabat numbering system) and the third constant domain CH3 (residues 341 -447).

[0060] By engineered Fc polypeptide, engineered Fc region and engineered Fc as the terms are interchangeably used herein, is meant an Fc polypeptide, or portion thereof, comprising at least one mutation, e.g., an amino acid substitution, introducing a site for conjugation. Preferably, the mutation introduces a cysteine in place of the naturally-occurring amino acid residue at that position, where the mutation creates a reactive site (e.g., a reactive sulfhydryl group) for conjugation of a moiety to the Fc.

[0061] The term monoclonal antibody or mAb refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Preferably, a monoclonal antibody of the invention exists in a homogeneous or substantially homogeneous population. Humanized antibody refers to forms of non-human (e.g. rat) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.13DM2\301045646.1PATENT Docket No. Y7969-99073

[0062] Human antibody or fully human antibody refers to those antibodies derived from transgenic mice carrying human antibody genes or from human cells.

[0063] The term chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a rat antibody and the constant region sequences are derived from a human antibody.

[0064] A therapeutic agent is an agent that exerts a cytotoxic, cytostatic, and / or immunomodulatory effect on cancer cells or activated immune cells. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cytostatic agents, and immunomodulatory agents.

[0065] An antibody, an antibody conjugate, or a polypeptide that preferentially binds or specifically binds (used interchangeably herein) to a target is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit specific binding or preferential binding if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody specifically binds or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. It is also understood that by reading this definition, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. ECso is a measurement of binding capacity and is defined as the half maximal effective concentration of an antibody or antibody-drug conjugate that is needed to produce a response halfway between the baseline and maximum.

[0066] The term “apex epitope” refers to a quaternary epitope formed at the trimer apex of the HIV envelope protein, comprising regions from the V1 / V2 domains. This epitope is specifically targeted by certain classes of bnAbs including PCT64-class, PG9-class, and CH01-04-class antibodies.14DM2\301045646.1PATENT Docket No. Y7969-99073

[0067] As used herein, the term “HCDR3” refers to the third complementarity determining region of the antibody heavy chain. A “long HCDR3” refers to an HCDR3 sequence of 24 amino acids or greater in length, which is characteristic of many apex-targeting bnAbs.

[0068] The term “DDY motif’ refers to a specific amino acid sequence comprising aspartic acid-aspartic acid-tyrosine, which plays a crucial role in apex epitope recognition by certain antibodies. This motif may be encoded directly by germline genes (e.g., IGHD3-15) or arise through V(D)J recombination or somatic hypermutation.

[0069] Self-assembling nanoparticle” refers to a structure composed of multiple protein subunits that spontaneously assemble into a defined, nanoscale particle. HIV Env trimers may be fused to self-assembling nanoparticles such as ferritin, lumazine synthase, and 153-50 nanoparticles to enhance immunogenicity by displaying multiple copies of the Env trimer in an organized array.

[0070] The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence” are used interchangeably to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. These terms also encompass proteins modified naturally or by intervention, including but not limited to disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0071] A “conservative amino acid change” refers to replacement of an amino acid residue with one having a similar side chain. Families of amino acid residues having similar side chains include:• Basic side chains (lysine, arginine, histidine)• Acidic side chains (aspartic acid, glutamic acid)• Non-charged polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine)• Non-polar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan)• Beta-branched side chains (threonine, valine, isoleucine)• Aromatic side chains (tyrosine, phenylalanine, tryptophan, histidine)15DM2\301045646.1PATENT Docket No. Y7969-99073

[0072] As used herein, the terms “env” or “envelope glycoprotein” refers to the surface protein of HIV responsible for binding to cellular receptors and mediating viral entry into host cells. In some embodiments, the Env protein may be engineered to form stabilized trimers or fused to nanoparticles to enhance immunogenicity.

[0073] A “conservative amino acid change” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid and glutamic acid), non-charged amino acids or polar side chains (e g. glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), beta-branched side chains (e.g. threonine, valine and isoleucine), and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan and histidine).

[0074] As used herein, the terms “antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and / or cellular type directed against that protein.

[0075] As used herein the terms “nucleotide sequences” and “nucleic acid sequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. The nucleic acid may be single-stranded, or partially or completely double-stranded (duplex). Duplex nucleic acids may be homoduplex or heteroduplex.

[0076] As used herein the term “transgene” may be used to refer to “recombinant” nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present invention. The term “recombinant” means a nucleotide sequence that has been manipulated “by man” and which does not occur in nature, or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man” means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc.16DM2\301045646.1PATENT Docket No. Y7969-99073

[0077] For example, in one embodiment the nucleotide sequences may be mutated such that the activity of the encoded proteins in vivo is abrogated. In another embodiment the nucleotide sequences may be codon optimized, for example the codons may be optimized for human use. In preferred embodiments the nucleotide sequences of the invention are both mutated to abrogate the normal in vivo function of the encoded proteins, and codon optimized for human use. For example, each of the sequences of the invention, such as the HIV Env proteins, may be altered in these ways.

[0078] As regards codon optimization, the nucleic acid molecules of the invention have a nucleotide sequence that encodes the antigens of the invention and may be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced. Many viruses, including HIV, use a large number of rare codons and, by altering these codons to correspond to codons commonly used in the desired subject, enhanced expression of the antigens may be achieved. In a preferred embodiment, the codons used are “humanized” codons, i.e., the codons are those that appear frequently in highly expressed human genes (Andre et al., J. Virol.72:1497-1503, 1998) instead of those codons that are frequently used by HIV. Such codon usage provides for efficient expression of the transgenic HIV proteins in human cells. Any suitable method of codon optimization may be used. Such methods, and the selection of such methods, are well known to those of skill in the art. In addition, there are several companies that will optimize codons of sequences, such as Geneart (geneart.com). Thus, the nucleotide sequences of the invention may readily be codon optimized.

[0079] The invention further encompasses nucleotide sequences encoding functionally and / or antigenically equivalent variants and derivatives of the antigens of the invention and functionally equivalent fragments thereof. These functionally equivalent variants, derivatives, and fragments display the ability to retain antigenic activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide. Conservative amino acid substitutions are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine; and phenylalanine / tyrosine / tryptophan. In one embodiment, the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at17DM2\301045646.1PATENT Docket No. Y7969-99073 least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide or a nucleotide sequence encoding the same of interest.

[0080] For the purposes of the present invention, sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.

[0081] Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.

[0082] Advantageous for use according to the present invention is the WU-BLAST (Washington University BLAST) version 2.0 software. WU-BLAST version 2.0 executable programs for several UNIX platforms may be downloaded from ftp: / / blast.wustl.edu / blast / executables. This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., Journal of Molecular Biology 1990, 215: 403-410; Gish & States, 1993, Nature Genetics 3: 266-272; and Karlin & Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein).

[0083] The various recombinant nucleotide sequences and immunogens of the invention are made using standard recombinant DNA and cloning techniques. Such techniques are well known to those of skill in the art. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989).18DM2\301045646.1PATENT Docket No. Y7969-99073

[0084] The nucleotide sequences of the present invention may be inserted into “vectors.” The term “vector” is widely used and understood by those of skill in the art, and as used herein the term “vector” is used consistent with its meaning to those of skill in the art. For example, the term “vector” is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.

[0085] Any vector that allows expression of the immunogen of the present invention may be used in accordance with the present invention. In certain embodiments, the immunogen of the present invention may be used in vitro (such as using cell-free expression systems) and / or in cultured cells grown in vitro in order to produce the encoded immunogens, which may then be used for various applications such as in the production of proteinaceous vaccines. For such applications, any vector that allows expression of the immunogens in vitro and / or in cultured cells may be used.

[0086] In certain aspects, the present invention provides expression vectors designed to optimize immunogen production while maintaining stability and high expression levels. In some embodiments, the expression vectors comprise multiple regulatory elements that facilitate protein expression in both research and manufacturing settings.

[0087] In one embodiment, the expression cassette includes a cytomegalovirus (CMV) immediate early promoter / enhancer region. In other embodiments, alternative promoters may be employed, including but not limited to the EFla promoter, CAG promoter, or tissue-specific promoters. In some aspects, the promoter region is followed by a synthetic intron, though in alternative embodiments, natural introns or other enhancing elements may be utilized.

[0088] In certain embodiments, the vector comprises a Kozak consensus sequence (GCCGCCACC) positioned upstream of the immunogen coding sequence. Alternative embodiments may employ modified Kozak sequences optimized for specific expression systems or cell types.

[0089] The immunogen coding sequence may be flanked by various regulatory elements. In some embodiments, the 5' untranslated region is engineered to eliminate alternative start codons. In certain aspects, the 3' untranslated region contains the bovine growth hormone (BGH) polyadenylation signal, while in other embodiments, alternative polyadenylation signals may be employed, such as the SV40 late poly(A) signal or synthetic poly(A) sequences.19DM2\301045646.1PATENT Docket No. Y7969-99073

[0090] In some embodiments, the vectors incorporate the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In alternative embodiments, other expressionenhancing elements may be utilized, including but not limited to chimeric introns or optimized nuclear export signals.

[0091] The vectors may include various selection markers. In one embodiment, an ampicillin resistance gene enables bacterial selection, while a neomycin resistance gene allows for mammalian cell selection. Alternative embodiments may employ different selection markers, such as kanamycin resistance, zeocin resistance, or auxotrophic markers.

[0092] In certain aspects, the complete expression cassette comprises the following elements in order from 5' to 3':- Enhancer / promoter region- Intron or other enhancing element- Kozak sequence or alternative translation enhancer- Start codon- Immunogen coding sequence- Stop codon- Post-transcriptional regulatory element- Polyadenylation signal

[0093] In some embodiments, for viral production applications, the expression cassette is incorporated into a modified lentiviral vector backbone. Alternative embodiments may employ other viral vectors, including but not limited to adenoviral vectors, AAV vectors, or alphavirus vectors.

[0094] In certain aspects, the vector backbone includes specific origins of replication. In one embodiment, the pUC origin enables high-copy bacterial amplification, while in other embodiments, alternative origins such as pl5A or pBR322 may be employed for different copy number control.

[0095] For DNA vaccination applications, in some embodiments, the expression cassette is incorporated into a modified pVAXl backbone. Alternative embodiments may employ other DNA vaccine vectors that meet regulatory requirements while offering specific advantages for particular applications.20DM2\301045646.1PATENT Docket No. Y7969-99073

[0096] In certain embodiments, the vectors are designed with specific restriction sites flanking key elements. Common restriction sites may include, but are not limited to, Nhel, Xbal, Notl, and Xhol. Alternative embodiments may employ different restriction sites or utilize restriction-free cloning methods.

[0097] The vectors described herein may undergo various stability tests. In some embodiments, testing includes multiple freeze-thaw cycles, extended storage at different temperatures, and passage stability studies. Alternative stability testing protocols may be employed depending on specific regulatory requirements or application needs.

[0098] In particular embodiments, the vectors are verified through sequencing. In some aspects, this includes comprehensive coverage of junction regions and regulatory elements, while in other embodiments, alternative verification methods may be employed, such as restriction mapping or functional assays.

[0099] In some embodiments, a host cell described herein is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER. C6, HEK-293T, NIH-3T3, HeLa, BHK, HepG2, SP2 / 0, Rl.l, B-W, L-M, COS-1, COS-7, BSC1, BSC40, BMT10, plant cell, insect cell, and human cell in tissue culture.

[0100] Examples of suitable mammalian host cell lines include but are not limited to CHO, VERO, BHK, HeLa, MDCK, HEK-293T, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e g., COS-1 or COS-7), PER. C6, HsS78Bst, HepG2, SP2 / 0, Rl.l, B-W, L-M, BSC1, BSC40, BMT10 cells, A549 cells, Jurkat cells, Renka cells, CT26 cells, PC-12 cells, TZM-bl cells, MT-4 cells, H9 cells, and primary human peripheral blood mononuclear cells (PBMCs).

[0101] For applications where it is desired that the immunogens be expressed in vivo, for example when the transgenes of the invention are used in DNA or DNA-containing vaccines, any vector that allows for the expression of the antibodies of the present invention and is safe for use in vivo may be used. In preferred embodiments, the vectors used are safe for use in humans, mammals and / or laboratory animals.

[0102] For the immunogens of the present invention to be expressed, the protein coding sequence should be “operably linked” to regulatory or nucleic acid control sequences that direct transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid21DM2\301045646.1PATENT Docket No. Y7969-99073 control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the nucleic acid control sequence. The “nucleic acid control sequence” may be any nucleic acid element, such as, but not limited to promoters, enhancers, internal ribosome entry site (IRES), introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. For VSV, the gene also can be operably linked to intergenic regions that control gene expression.

[0103] The vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or intergenic region, such that the immunogen of the invention may be expressed.

[0104] As used herein, mutations that facilitate proper protein folding refer to modifications that improve the expression, processing, or conformational integrity of the Env polypeptide. Such modifications include replacement of the native Env signal peptide with heterologous leader sequences, for example the CD5 or tissue plasminogen activator (tPA) leaders, and in some expression contexts other well-characterized leaders such as Gaussia luciferase or interleukin-2, to enhance secretion and transit through the secretory pathway. In constructs designed to be cleaved, enhancement of the native REKR furin site with a polybasic sequence, for example RRRRRR, can improve precursor processing. In uncleaved, “native flexibly linked” trimers, the REKR site can instead be replaced by short flexible linkers, for example two or three repeats of a Gly4-Ser motif, to covalently join gpl20 and gp41 while allowing native-like association of the subunits. Additional modifications that improve folding and translation include use of human-codon-optimized nucleotide sequences to mitigate ribosomal pausing. Glycan engineering can also influence folding and aggregation in a strain- and site-dependent manner; for example, adjustments at the N276 site in gpl20 or at selected V4 / V5 sites can alter processing or conformational sampling, whereas other glycans are essential for proper folding and subunit association. In some designs, adding glycans to mask off-target hydrophobic patches can reduce exposure of non-native surfaces during biosynthesis.

[0105] As used herein, mutations that enhance trimer stability refer to amino acid substitutions and structural modifications that favor a stable, prefusion closed Env trimer. Stabilizing designs include introduction of a gpl20-gp41 disulfide bridge, for example A501C in gpl20 paired with T605C in gp41 (the “SOS” change), together with a helix-breaking substitution in gp41 heptad22DM2\301045646.1PATENT Docket No. Y7969-99073 repeat 1 (HR1), for example I559P, to impede premature transition of HR1 toward the postfusion state. Additional disulfides can further constrain conformational transitions, for example an intra-gpl20 “DS” disulfide between I201C and A433C that reduces CD4-induced opening, or engineered cysteine pairs such as A73C in gpl20 combined with A561C in gp41 to stabilize the trimer base. Further stabilization can be achieved with substitutions that improve the packing and dynamics of the trimer core, including, by way of example, HR1-region changes such as L568D, V570H, or T569G, and gp41 core or fusion-peptide-proximal changes such as A561P, F519S, or R585H. In addition, substitutions that reduce apex opening and V3 exposure, such as H66R or E64K in gpl20 layer 1 combined with A316W at the V3 base, can shift the equilibrium toward the closed prefusion state. These and related sets of substitutions have been used in BG505-based backbones, including MD-series hyper-stabilized trimers, and can be applied in combination to reduce spontaneous sampling of downstream conformations while preserving neutralizing epitopes in the prefusion trimer.

[0106] Any suitable vector may be used depending on the application. For example, plasmids, viral vectors, bacterial vectors, protozoal vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, and the like, may be used. Suitable vectors may be selected by the skilled artisan taking into consideration the characteristics of the vector and the requirements for expressing the immunogens under the identified circumstances.

[0107] As used herein, the term “self-assembling nanoparticle” refers to a structure composed of multiple protein subunits that spontaneously assemble into a defined, nanoscale particle. In the context of this invention, HIV Env trimers may be fused to self-assembling nanoparticles to enhance their immunogenicity by displaying multiple copies of the Env trimer in an organized array. Examples of self-assembling nanoparticles include ferritin, lumazine synthase, and 153-50 nanoparticles, which can present the Env trimer in a geometrically optimized configuration, potentially enhancing B cell activation and boosting the immune response toward apex-targeting broadly neutralizing antibodies (bnAbs).

[0108] As used herein, the term “tissue-resident memory T cells” refers to a subset of memory T cells that remain within peripheral tissues, including mucosal surfaces, where they provide a rapid and robust immune response upon re-exposure to HIV antigens. These cells are capable of a fast and localized response, aiding in long-term immune protection following vaccination.23DM2\301045646.1PATENT Docket No. Y7969-99073

[0109] The term “pharmaceutical composition” is used herein to define a solid, liquid, or particulate composition in a form, concentration, and level of purity suitable for administration to a subject (e.g., a human patient). Upon administration, this composition may elicit the desired immune response against HIV. The terms “immunogenic composition,” “immunological composition,” and “immunogenic or immunological composition” cover any composition that stimulates an immune response against HIV, including compositions designed to elicit bnAbs that target conserved regions, such as the apex epitope of the HIV Env protein. Terms such as “vaccinal composition,” “vaccine,” and “vaccine composition” refer to any composition that induces a protective immune response against HIV, potentially through administration or injection, and provides efficacious protection by stimulating bnAbs or T cell responses to the virus. An immunogenic or immunological composition may induce a neutralizing antibody response and may be used in treating individuals exposed to HIV, promoting immune response priming, such as by targeting bnAb precursors that bind conserved HIV Env regions.

[0110] Modified HIV Envelope Protein

[0111] The present invention provides engineered immunogens comprising modified HIV envelope (Env) proteins designed to induce broadly neutralizing antibodies targeting the apex epitope. These modifications may include, but are not limited to:

[0112] (a) Mutations in the Apex region that enhance recognition by bnAb precursors; (b) Mutations that promote proper protein trimer folding and stability; (c) Strategic addition or removal of glycosylation sites; and (d) Combinations of any of the above modifications.

[0113] In some embodiments, mutations that promote proper protein trimer folding and stability may comprise mutations that enable disulfide bond formation, such as, for e.g., SOSIP mutations, i.e. mutations that introduce cysteine residues to form disulfide bonds between subunits; incorporating proline residues to rigidify flexible regions and stabilize the prefusion conformation of the protein; altering amino acids in the heptad repeat 1 (HR1) region to prevent undesirable conformational changes that lead to instability; adding or removing N-linked glycosylation sites to enhance proper folding and mask or expose specific epitopes important for immune recognition; attaching trimerization motifs like the GCN4 leucine zipper or the foldon domain to promote trimer formation and stability; modifying the furin cleavage site for efficient processing or replacing it with a flexible linker to maintain structural integrity; introducing amino acid changes that24DM2\301045646.1PATENT Docket No. Y7969-99073 strengthen inter-subunit interactions within the trimer; and removing flexible regions such as the VI V2 and V3 loops to reduce conformational heterogeneity and improve stability.

[0114] In certain embodiments, the modified Env protein comprises specific mutations in the V1 / V2 region, including P76S, T128G, and N195S mutations relative to the ApexGT5 sequence. These positions are numbered according to the HXB2 sequence:

[0115] AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPN PQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNIT DDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCN TSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQ LLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGD IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGE FFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVI RCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCK RRVVGRRRRRRAVAIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRA PEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSN RNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 4).

[0116] The positions of these mutations were selected based on structural analysis and their role in enhancing binding to bnAb precursors. The T128G mutation enhances binding to germline precursors. The P76S and N195S mutations stabilize V1V2 apex conformation.

[0117] The mutations P76S, T128G, and N195S were identified through structure-guided directed evolution using mammalian cell-surface display. This approach utilized two complementary libraries: a random mutagenesis library targeting the V1 / V2 region using error-prone PCR, and a combinatorial saturation mutagenesis library targeting specific residues within the PCT64 antigen-antibody binding interface. Selection was performed using the least mutated common ancestor of PCT64 (PCT64 LMCA) as a positive probe and the non-neutralizing antibody B6 as a negative probe, enabling identification of mutations that enhanced affinity to germline antibodies while maintaining proper trimerization.

[0118] Surface plasmon resonance (SPR) analysis demonstrated that ApexGT6 exhibits significantly enhanced binding to PG9 precursors compared to previous designs. When tested against Pre.15, a PG9-like NGS precursor, and Pre.7, a PCT64-like NGS precursor, ApexGT625DM2\301045646.1PATENT Docket No. Y7969-99073 showed improved affinity compared to MD39 and earlier ApexGT variants. This enhanced binding profile suggests successful optimization of the immunogen for engaging bnAb precursors.

[0119] In specific embodiments, glycosylation sites may be strategically introduced at positions N241 and N289. These modifications serve to minimize off-target responses to glycan holes outside the apex region. The combination of these glycosylation sites with the V1 / V2 mutations results in the ApexGT6 congly variant, which demonstrates improved binding characteristics compared to previous designs. Comprehensive glycan analysis using the DeGlyPHER method demonstrated that ApexGT6 maintains optimal occupancy at highly conserved glycosylation sites within the apex region. The glycosylation profile showed proper complex glycan formation at key positions, with particular attention to the N156 and N160 glycans that allow for apex epitope formation. This analysis confirmed successful engineering of the glycan shield while maintaining accessibility of key binding surfaces.

[0120] The sequence of ApexGT6 congly is:

[0121] AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVSTDPN PQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVGLQCTNVTNNIT DDMRGELKNCSFNATTELRNKRQKVYSLFYRLDIVPMVDLWTNYRLISCNTSAITQACP KVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLA EEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQA HCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTS GLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVAIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 5).

[0122] Analysis of the V2b loop region revealed important structural features that influence antibody recognition. Natural HIV isolates typically display V2b loops ranging from 13 to 16 amino acids in length, with one or two N-linked glycosylation sites. These loops are predominantly hydrophilic, contrasting with the engineered hydrophobic mutations in ApexGT6 that enhance initial precursor binding. This understanding of native V2b characteristics guided the development of variant immunogens used to assess antibody maturation toward recognition of more native-like epitopes.26DM2\301045646.1PATENT Docket No. Y7969-99073

[0123] For membrane-bound applications, the invention provides ApexGT6 LI 4, engineered as a cleavage-independent version capable of being expressed as a membrane-bound trimer (gpl 51). The sequence of ApexGT6 L14 gpl 51 is:

[0124] AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVSTDPN PQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVGLQCTNVTNNIT DDMRGELKNCSFNATTELRNKRQKVYSLFYRLDIVPMVDLWTNYRLISCNTSAITQACP KVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAE EEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAH CNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSG LFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGL ILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSG SGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAP EPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNR NLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDI SNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 6).

[0125] Protein Characteristics and Structure

[0126] The modified Env proteins of the invention demonstrate several advantageous characteristics:

[0127] 1 Enhanced Affinity for bnAb Precursors: Surface plasmon resonance (SPR) analysis demonstrates significantly improved binding to both PCT64 and PG9 precursors identified through next-generation sequencing of HIV seronegative individuals. The improvements in affinity include:• Enhanced binding to PG9-like NGS precursor (Pre.15)• Improved affinity for PCT64-like NGS precursor (Pre.7)• Consistent recognition of germline-reverted antibody variants

[0128] 2. Structural Stability: Differential scanning calorimetry analysis reveals excellent thermal stability with specific melting temperatures:• ApexGT5: 72.36°C• ApexGT5.congly: 72.58°C• ApexGT6: 71.48°C• ApexGT6.congly: 70.91°C27DM2\301045646.1PATENT Docket No. Y7969-99073

[0129] 3. Glycosylation Profile: Comprehensive glycosylation analysis using the DeGlyPHER method demonstrates:• Proper occupancy at highly conserved glycosylation sites within the apex region • Strategic placement of glycans to shield non-neutralizing epitopes• Maintenance of key gly can-dependent epitope structures

[0130] 4. Antigenic Properties: The modified proteins maintain proper antigenic profiles as demonstrated by:• Strong binding to trimer-specific antibodies• Minimal reactivity with non-neutralizing epitopes• Preserved recognition of apex-specific bnAbs

[0131] Production Methods

[0132] The modified HIV Env proteins of the present invention can be produced through various expression systems and methods. In one embodiment, the proteins are expressed in mammalian cells, particularly HEK293F cells cultured in FreeStyle media. For soluble trimers, the expression constructs typically comprise the modified Env sequence co-transfected with furin in a 2:1 ratio using polyethylenimine (PEI) as a transfection reagent in Opti-MEM™ reduced serum medium. The proteins are harvested from the supernatant after seven days of incubation at 37°C.

[0133] Purification of the expressed proteins may be accomplished through multiple chromatographic steps. For non-tagged trimers, initial purification utilizes lectin affinity chromatography, typically employing 7.5 mL of lectin beads per IL of protein supernatant. The trimer fractions are further isolated using size exclusion chromatography (SEC) on a Superdex 200 PG SEC column. For his-tagged trimers, the purification process involves Ni++ affinity chromatography followed by SEC. When biotinylated probes are required, the proteins are expressed with both his-tag and avi-tag modifications, purified by Ni++ affinity chromatography and SEC, then biotinylated using a BirA biotin-protein ligase reaction kit according to manufacturer's instructions.

[0134] The oligomeric state and structural integrity of the purified trimers can be verified through size exclusion chromatography-multi-angle light scattering using the DAWN HELEOS II system with Optilab T-rEX refractometer. This analysis confirms the proper assembly and stability of the trimeric structure, which is essential for maintaining the quaternary epitopes targeted by apex bnAbs.28DM2\301045646.1PATENT Docket No. Y7969-99073

[0135] mRNA Production and Formulation

[0136] For mRNA-based delivery, the modified Env sequences are encoded in messenger RNA produced through in vitro transcription. The mRNA is typically synthesized using a DNA template containing the appropriate regulatory elements, including a 5' cap structure and 3' poly(A) tail. The resulting mRNA can be purified using established methods such as chromatographic separation or precipitation techniques.

[0137] The purified mRNA is then formulated into lipid nanoparticles (LNPs) to protect the mRNA from degradation and facilitate cellular uptake. The LNP formulation typically comprises a combination of ionizable lipids, helper lipids, cholesterol, and PEG-modified lipids. The specific composition and ratio of lipid components can be optimized to achieve desired biodistribution and expression characteristics.

[0138] Characterization Methods

[0139] Comprehensive characterization of the modified Env proteins involves multiple complementary analytical techniques. Surface plasmon resonance (SPR) analysis is conducted using a Carterra LSA system with CMDP Sensor Chips and HBS-EP+ running buffer supplemented with BSA. This method enables detailed kinetic analysis of antibody-antigen interactions, with capture antibodies typically immobilized at concentrations of 25 pg / mL with 10 minutes contact time. The analysis includes both double referencing with interspot and blank controls, and utilizes the Langmuir model for binding kinetics determination.

[0140] Antigenic profiling through enzyme-linked immunosorbent assay (ELISA) provides crucial information about epitope accessibility and proper protein folding. The assays are typically performed using 96-well plates coated with 6x-His (SEQ ID NO: 18) Epitope Tag Rabbit Antibody at 2 pg / mL in PBS. After blocking with 5% milk and 1% FBS in PBS-T, purified His-tagged trimers are added at 2 pg / mL, followed by serial dilutions of antigenic profiling mAbs in 1% FBS PBS-T.

[0141] Advanced Characterization Methods

[0142] Differential scanning calorimetry (DSC) provides essential information about the thermal stability and structural integrity of the modified Env proteins. These experiments are performed using a MicroCai VP-Capillary differential scanning calorimeter, with samples diluted in HBS buffer to 0.25 mg / mL. The system equilibrates at 20°C for 15 minutes before heating to 90°C at a rate of 90°C / h. Analysis of the resulting thermograms involves buffer correction,29DM2\301045646.1PATENT Docket No. Y7969-99073 normalization, and baseline subtraction, with data fitting performed using a non-two-state model to accurately represent the complex unfolding behavior of these multimeric proteins.

[0143] The glycosylation profile of the modified Env proteins represents an aspect of their immunogenic properties. Comprehensive glycan analysis is performed using the DeGlyPHER method, which enables precise determination of site-specific glycan occupancy and processivity. This process begins with protein reduction using TCEP-HC1 and alkylation with 2-Chloroacetamide in ammonium acetate buffer. Initial deglycosylation is performed using Endo H, followed by Proteinase K digestion and subsequent deglycosylation steps. The resulting peptides are analyzed by LC-MS / MS using a Q Exactive HF-X mass spectrometer, with separation performed on a 25 cm column packed with BEH 1.7 pm Cl 8 resin.

[0144] Mass spectrometry data acquisition employs a sophisticated data-dependent mode, with full MS 1 scans collected at 120,000 resolution. The ten most abundant ions per scan undergo HCD MS / MS analysis at 25 NCE, with dynamic exclusion enabled and singly charged ions excluded. Data processing utilizes the Integrated Proteomics Pipeline, incorporating RawConverter for spectra extraction and ProLuCID for database searching against a comprehensive protein database including the UniProt reviewed proteome for Homo sapiens and relevant enzyme sequences.

[0145] Immunological Characterization

[0146] The immunological properties of the modified Env proteins are extensively characterized through a combination of cellular and molecular approaches. Flow cytometry analysis employs a sophisticated multi-parameter strategy to identify and isolate Apex-specific B cells. For germinal center B cells, analysis focuses on CD20+CD38‘ populations, while memory B cells are identified as CD20+IgD'. Epitope specificity is determined through differential binding analysis using fluorophore-conjugated probes of both the wild-type immunogen and knockout variants containing mutations that abrogate apex epitope recognition.

[0147] The preparation of flow cytometry probes requires careful attention to maintain protein integrity and specificity. Biotinylated proteins are complexed with fluorophore-conjugated streptavidin in a stepwise manner, with additions made in 1 / 3 increments over 45 minutes at room temperature. This approach ensures proper probe formation while minimizing protein aggregation or denaturation. Staining protocols typically involve sequential incubation with different probes at 4°C, with appropriate washing steps between additions to minimize background and ensure specificity.30DM2\301045646.1PATENT Docket No. Y7969-99073

[0148] Single-cell analysis of B cell responses provides detailed information about the molecular characteristics of the induced immune response. This involves isolation of individual B cells through fluorescence-activated cell sorting, followed by sequencing of paired heavy and light chain genes. The analysis pipeline incorporates custom primers designed to target rhesus macaque BCR constant regions, with specific modifications to accommodate the unique aspects of the macaque immune system.

[0149] B Cell Response Analysis

[0150] Analysis of B cell responses following immunization requires sophisticated molecular and cellular techniques to fully characterize the induced immune response. The sequencing of B cell receptors (BCRs) begins with the isolation of single cells using index sorting on a FACSymphony S6 flow cytometer. For lymph node fine-needle aspirates (FNA) samples, indexed V(D)J, Feature Barcode, and GEX libraries are prepared according to the Single Indexed 10X Genomics V(D)J 5' v.1.1 protocol with Feature barcoding modifications. Peripheral blood mononuclear cell (PBMC) samples undergo similar processing using the Dual Indexed 10X Genomics V(D)J 5' v.2 protocol with appropriate Feature barcoding adaptations.

[0151] The sequencing strategy employs custom-designed primers specifically targeting rhesus macaque BCR constant regions. The primer design incorporates multiple forward and reverse sequences to ensure comprehensive coverage of the repertoire. Forward primers typically contain the sequence AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA CGCTC (SEQ ID NO: 7), while reverse primers comprise a set of carefully selected sequences including AGGGCACAGCCACATCCT SEQ ID NO: 8), TTGGTGTTGCTGGGCTT SEQ ID NO: 9), TGACGTCCTTGGAAGCCA (SEQ ID NO: 10), and others optimized for the rhesus macaque immune system. These primers are used at specific concentrations, with forward primers at 1 µM and reverse primers at 0.5 µM per 100 µl PCR reaction.

[0152] Sequence Analysis and Repertoire Characterization

[0153] The analysis of sequencing data employs a sophisticated bioinformatics pipeline beginning with the assembly of full-length V(D)J reads using CellRanger v.3.0. This process incorporates a custom Rhesus Macaque germline VDJ library and includes modifications to the constants.py file to accommodate extended CDR3 lengths up to 110 nucleotides. Gene expression quantification utilizes the Ensemble MmullO genome assembly, with special consideration given to mitochondrial genes from Mmul9.31DM2\301045646.1PATENT Docket No. Y7969-99073

[0154] Data processing continues with the parsing of sequences into AIRR format using the Change-O pipeline from the Immcantation portal. This standardized format enables comprehensive analysis of the immune repertoire, including determination of clonal relationships, identification of somatic hypermutation patterns, and tracking of lineage development. The SADIE 0.4.31 library, employed with the “macaque” option, provides additional annotation capabilities specifically tailored to rhesus macaque sequences.

[0155] Antibody Characterization and Functional Analysis

[0156] The functional characterization of induced antibodies involves multiple complementary approaches to assess binding properties, neutralization capacity, and structural features. Surface plasmon resonance experiments, conducted on the Carterra LSA platform, provide detailed kinetic information about antibody-antigen interactions. These measurements employ CMDP Sensor Chips with carefully optimized running conditions using HBS-EP+ buffer supplemented with BSA at 1 mg / ml.

[0157] The preparation of capture surfaces requires precise control of coupling chemistry conditions. The concentration of activation reagents plays a crucial role, with optimal results achieved using modified concentrations of NHS (1.15 mg / ml) and EDC (7.5 mg / ml) during surface preparation. The capture antibody, typically sourced from SouthernBiotech, is applied at 25 pg / ml with a 10-minute contact time. Regeneration between binding cycles employs 1.7% phosphoric acid with 60-second contact times, applied in triplicate to ensure complete surface regeneration.

[0158] Structural Analysis of Antibody-Antigen Complexes

[0159] The structural characterization of antibody-antigen complexes provides crucial insights into the molecular basis of recognition and neutralization. Cryo-electron microscopy studies of antibody-antigen complexes require careful sample preparation and sophisticated data collection protocols. Complex formation typically involves overnight incubation of purified antibody Fabs with the trimeric immunogen at specifically optimized ratios. For example, preparation of the RM038-ApexGT6 complex involves combining 200 pg of ApexGT6 trimer with 300 pg of RM038 Fabs and RM20A3 Fabs, followed by size exclusion chromatography purification to isolate the desired complexes.

[0160] Sample vitrification employs a Vitrobot Mark IV operating under precisely controlled conditions, with the chamber temperature maintained at 10°C and humidity at 100%. Quantifoil Cu 1.2 / 1.3 300C-mesh grids undergo glow discharge treatment for 25 seconds at 15 mA using a32DM2\301045646.1PATENT Docket No. Y7969-99073 PELCO easiGLOW system immediately before sample application. The addition of lauryl maltose neopentyl glycol (LMNG) to a final concentration of 0.005 mM helps optimize sample distribution and ice quality. Blot times are carefully optimized between 5-7 seconds before plunge freezing into liquid ethane cooled by liquid nitrogen.

[0161] Data collection utilizes a 200 kV Glacios microscope equipped with a Falcon 4 detector, with automated data collection managed through EPU software. Image processing employs CryoSPARC Live for initial preprocessing, followed by comprehensive analysis in CryoSPARC 3.2 and 4.4. Particle selection combines both blob and template picking approaches, with iterative 2D classification steps to remove suboptimal particles. Initial volume generation uses Ab Initio procedures in CryoSPARC, followed by multiple rounds of refinement including homogeneous, non-uniform, and local refinement steps to achieve high-resolution reconstructions.

[0162] Antigenic Profiling and Epitope Mapping

[0163] Comprehensive antigenic profiling of the modified immunogens employs multiple complementary approaches. Cell surface antigenic profiling provides crucial information about the presentation of epitopes in a membrane context. This involves transfection of DNA-encoded membrane-bound trimers into HEK293F suspension cells using 293fectin Transfection Reagent, followed by a two-day incubation period at 37°C with constant agitation at 125rpm. The analysis employs a panel of well-characterized antibodies including trimer-specific bnAbs targeting various epitopes (interface / FP: PGT151, Apex: PGT145, PG9, and PCT64.35S) and non-neutralizing antibodies (V3: 4025, CD4bs: B6 and F105).

[0164] Flow cytometric analysis of cell surface expression and antibody binding utilizes a NovoCyte 3000 with NovoSampler Pro system, typically collecting data from approximately 50,000 live cells per condition. Cell preparation involves careful attention to maintaining cell viability and surface protein integrity throughout the staining process. Antibody solutions are prepared at 10 ug / mL in FACS buffer comprising HBSS supplemented with 1 mM EDTA and 1% BSA. Staining protocols incorporate appropriate dead cell exclusion dyes and secondary detection reagents optimized for signal-to-noise ratio.

[0165] Negative-stain electron microscopy polyclonal epitope mapping (nsEMPEM) provides detailed information about the distribution of antibody responses across the immunogen surface. This technique begins with the purification of IgG from plasma samples using an AKTA Pure system equipped with a HiTrap MabSelect PrismA column, followed by controlled papain33DM2\301045646.1PATENT Docket No. Y7969-99073 digestion to generate Fab fragments. Complex formation between isolated Fabs and trimeric immunogens requires careful optimization of conditions and ratios to achieve appropriate sample distribution for imaging.

[0166] Therapeutic Applications and Administration Methods

[0167] The immunogens of the present invention can be administered through multiple routes and formulations, each offering distinct advantages for specific applications. The adjuvanted protein formulation comprises the modified Env protein combined with saponin / MPLA nanoparticle (SMNP) adjuvant. An embodiment utilizes 50 pg of soluble ApexGT6 congly protein combined with 375 pg of SMNP adjuvant per administration site. For bilateral administration, this translates to a total dose of 100 pg protein and 750 pg adjuvant per immunization.

[0168] The mRNA-LNP formulation, comprising ApexGT6 L14 gpl51, provides an alternative delivery approach with unique advantages for protein expression and immune response characteristics. A typical dose comprises 50 pg of mRNA encoding the membrane-bound ApexGT6 formulated within lipid nanoparticles optimized for delivery to antigen-presenting cells. The LNP formulation is designed to protect the mRNA from degradation and facilitate cellular uptake, with the specific lipid composition optimized for delivery to antigen-presenting cells. Administration is typically performed via intramuscular injection, with bilateral deltoid administration providing optimal distribution of the immunogen.

[0169] Administration may be accomplished orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular (IM), intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. In an embodiment, IM is preferred, but other routes can be used such as subcutaneous or application to mucosal surfaces in the nose or mouth.

[0170] In an advantageous embodiment, the administration is IM. The dosage is measured in PFUs. The present invention illustrates that low doses of the vaccine are as effective as higher doses. The dosage administration may be about 102-107PFUs. Advantageously, the dosage may be about 104, 105, 106PFUs.

[0171] It is noted that humans may require higher vaccine dosages than mice or other experimental animals to elicit an effective immune response. In an aspect, the doses may be single doses or multiple doses over a period of time, in some aspects, single doses are preferred. Thus, one may scale up from animal experiments, e.g., rats, mice, and the like, to humans, by techniques34DM2\301045646.1PATENT Docket No. Y7969-99073 from this disclosure and documents cited herein and the knowledge in the art, without undue experimentation.

[0172] When administering a therapeutic of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier may be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0173] Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the vaccine preparation. Formulations that stabilize the virus are also contemplated. Additions such as, but not limited to, carbohydrates (such as sucrose or trehalose), gelatin, hydrolyzed gelatin, amino acids, etc. are contemplated.

[0174] Sterile injectable solutions may be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate buffered solution with various amounts of the other ingredients, as desired.

[0175] A pharmacological formulation of the present invention, e.g., which may comprise a therapeutic compound or polypeptide of the present invention, may be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the compounds utilized in the present invention may be administered parenterally to the patient in the form of or polymer matrices, liposomes, and microspheres.

[0176] Adjuvants are any substance whose admixture with an administered antigen increases or otherwise modifies the immune response to said antigen. Adjuvants may for example be selected from the group consisting of A1K(SO4)2, AlNa(SC>4)2, A1NH4 (SO4), silica, alum, Al(0H)3, Cas35DM2\301045646.1PATENT Docket No. Y7969-99073 (PC>4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'2'-dipalmitoyl-s- n-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene / Tween-80. RTM emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see U. S. Pat. Nos. 58,767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21. Preferred adjuvants to be used with the invention include Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants.

[0177] A pharmacological formulation of the compound and composition which may comprise an immunogen utilized in the present invention may be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques, which deliver the compound orally or intravenously and retain the biological activity, are preferred.

[0178] In one embodiment, a formulation of the present invention may be administered initially, and thereafter maintained by further administration. For instance, a formulation of the invention may be administered in one type of composition and thereafter further administered in a different or the same type of composition. For example, a formulation of the invention may be administered by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition, may be used. In the instance of a vaccine composition, the vaccine may be administered as a single dose, or the vaccine may incorporate set booster doses.

[0179] The quantity to be administered will vary for the patient being treated and whether the administration is for treatment or prevention and will vary from about 102-107plaque-forming units (PFUs) of the vaccine. The administering may be about 104PFU, about 105PFU or about 106PFU of the vaccine.36DM2\301045646.1PATENT Docket No. Y7969-99073

[0180] Of course, for any composition to be administered to an animal or human, including the components thereof, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LDso in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response, such as by titrations of sera and analysis thereof for antibodies or antigens, e.g., by ELISA and / or Rapid Fluorescent Foci Inhibition Test (RFFIT) analysis. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations may be ascertained without undue experimentation. For instance, dosages may be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Thus, the skilled artisan may readily determine the amount of compound and optional additives, vehicles, and / or carrier in compositions and to be administered in methods of the invention. Typically, an adjuvant or additive is commonly used as 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations may be ascertained without undue experimentation.

[0181] Examples of compositions which may comprise a therapeutic of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as " REMINGTON'S PHARMACEUTICAL SCIENCE",37DM2\301045646.1PATENT Docket No. Y7969-99073 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0182] Compositions of the invention, are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the invention may be in the “solid” form of pills, tablets, capsules, caplets and the like, including “solid” preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. If nasal or respiratory (mucosal) administration is desired, compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers may preferably dispense a metered dose or, a dose having a particular particle size.

[0183] Compositions of the invention may contain pharmaceutically acceptable flavors and / or colors for rendering them more appealing, especially if they are administered orally. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like (e.g., for transdermal administration) and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2,500 to 6,500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity preferably of 2,500 to 5,000 cps, since above that range they become more difficult to administer. However, above that range, the compositions may approach solid or gelatin forms, which are then easily administered as a swallowed pill for oral ingestion.

[0184] Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection or orally. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.

[0185] Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form).38DM2\301045646.1PATENT Docket No. Y7969-99073

[0186] Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the active compound. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and / or flavors may also be present. The compositions may be isotonic, i.e., it may have the same osmotic pressure as blood and lacrimal fluid.

[0187] The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

[0188] Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0189] A pharmaceutically acceptable preservative may be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.

[0190] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert with respect to the active compound. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems may be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0191] It is generally envisaged that compounds and compositions of the invention will be administered by injection, as such compounds are to elicit bnAbs againsg HIV, and the skilled artisan may, from this disclosure and the knowledge in the art, formulate compounds and39DM2\301045646.1PATENT Docket No. Y7969-99073 compositions identified by herein methods for administration by injection and administer such compounds and compositions by injection.

[0192] The inventive compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally, the pH may be from about 3 to 8.5. Compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals may be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0193] Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0194] Dosing Regimens and Immunization Schedules

[0195] The timing of immunizations plays a crucial role in developing effective immune responses. A primary immunization schedule comprises an initial dose at day 0 followed by a boost immunization at week 8. This timing allows for the establishment and maturation of germinal center responses before secondary antigenic stimulation. The interval between doses may be adjusted based on monitoring of immune responses, with flexibility to modify the schedule according to observed B cell dynamics and antibody development.

[0196] Monitoring of immune responses typically involves collection of blood samples and, where feasible, fine-needle aspiration (FNA) of axillary lymph nodes at defined timepoints throughout the immunization schedule. Blood sampling enables tracking of serum antibody responses and circulating memory B cells, while FNA samples provide crucial information about germinal center dynamics and the evolution of antibody responses at the site of immune response generation.

[0197] Manufacturing Considerations40DM2\301045646.1PATENT Docket No. Y7969-99073

[0198] Production of the immunogens for clinical application requires careful attention to manufacturing processes and quality control. For protein-based immunogens, the manufacturing process begins with establishment of stable cell lines expressing the modified Env protein. Cell line development typically employs HEK293F cells adapted to suspension culture in chemically defined, serum-free medium. The expression system incorporates optimized signal sequences and regulatory elements to maximize protein production while maintaining proper folding and post-translational modifications.

[0199] The purification process for clinical-grade material involves multiple chromatographic steps designed to ensure both purity and proper protein conformation. Initial capture typically employs lectin affinity chromatography, followed by additional purification steps that may include ion exchange chromatography, size exclusion chromatography, and specific affinity steps depending on the protein construct. Each step is optimized to maintain trimer stability while removing process-related impurities and potential contaminants.

[0200] For mRNA production, manufacturing begins with generation of linear DNA templates through PCR or restriction enzyme digestion of plasmid DNA. In vitro transcription reactions are performed under carefully controlled conditions to ensure consistency in mRNA quality and yield. The transcription reaction typically includes modified nucleosides to reduce immunogenicity and improve stability. Purification of the mRNA product employs chromatographic techniques optimized for large-scale production while maintaining RNA integrity.

[0201] Quality Control and Analytical Methods

[0202] The implementation of a comprehensive quality control program is essential for ensuring consistent production of immunogens that meet predetermined specifications. Analytical ultracentrifugation serves as a primary tool for assessing trimer stability and proper assembly of the modified Env proteins. In some aspects, these analyses are conducted using a Beckman XL-A ultracentrifuge with scanning UV / Vis optics, which enables continuous monitoring of protein distribution throughout the sedimentation process. In some aspects, to generate a complete understanding of protein behavior in solution, multiple experimental conditions are employed, varying both rotor speeds and protein concentrations across a defined range.

[0203] Glycan analysis represents an critical aspect of quality control, given the fundamental role of glycosylation in immunogen function and recognition. In some aspects, the analytical process begins with careful enzymatic release of N-linked glycans from the protein surface,41DM2\301045646.1PATENT Docket No. Y7969-99073 followed by specific fluorescent labeling procedures optimized for subsequent analytical separation. In an aspect, the characterization employs hydrophilic interaction liquid chromatography (HILIC) directly coupled to mass spectrometric analysis, providing detailed information about glycan structures and their distribution patterns across the various glycosylation sites on the immunogen surface. In an aspect, this analysis allows for monitoring the glycosylation status at positions known to influence recognition and binding by broadly neutralizing antibodies.

[0204] Stability Testing and Storage Conditions

[0205] The development of a robust stability testing program provides crucial information about product integrity under various storage and handling conditions. Real-time stability studies are conducted at the intended storage temperature of -80°C for protein-based immunogens and -20°C for mRNA formulations, with regular testing intervals established to monitor product quality over time. Accelerated stability studies, conducted at elevated temperatures, provide additional information about potential degradation pathways and help establish product shelf life. The stability program incorporates comprehensive freeze-thaw studies to determine the impact of temperature cycling on product quality, as multiple freeze-thaw cycles may be necessary during product handling and administration.

[0206] Photostability testing examines the sensitivity of the immunogens to light exposure, with samples exposed to defined light conditions according to ICH guidelines. These studies inform proper handling and storage requirements, particularly regarding protection from light during manufacturing, storage, and administration. Container closure integrity testing ensures that the primary packaging maintains its protective function throughout the product shelf life, preventing microbial contamination and maintaining product sterility.

[0207] Regulatory Considerations and Documentation

[0208] The development of HIV immunogens requires extensive regulatory documentation covering all aspects of product development and manufacturing. The chemistry, manufacturing, and controls documentation package provides comprehensive information about raw material specifications, including detailed requirements for materials such as cell culture components, purification resins, and excipients used in final formulation. Process validation studies demonstrate the consistency and reliability of the manufacturing process, with particular attention paid to process parameters that may impact product quality.

[0209] Clinical Trial Design Considerations42DM2\301045646.1PATENT Docket No. Y7969-99073

[0210] The evaluation of HIV immunogens in clinical settings requires careful attention to trial design and implementation. Initial safety and immunogenicity studies typically employ a dose-escalation design beginning with a conservative dose level determined from preclinical studies. The standard immunization regimen involves bilateral administration in the deltoid region, with precise specifications for injection technique and volume. For protein immunizations, the injection sites must be separated by sufficient distance to enable independent evaluation of local reactions, while ensuring proper distribution of the immunogen and adjuvant combination.

[0211] The timing of sample collection represents an aspect of clinical trial design, with timepoints selected to capture both early and late immune responses. Blood samples are typically collected pre-immunization and at defined intervals post-immunization, with particular attention to timepoints corresponding to expected peak germinal center activity and memory B cell formation. When feasible, fine-needle aspiration of draining lymph nodes provides valuable information about the development and evolution of germinal center responses, though this procedure requires specifically trained personnel and appropriate facilities.

[0212] Safety Monitoring and Assessment

[0213] The implementation of a comprehensive safety monitoring program encompasses both immediate and long-term follow-up of study participants. Local injection site reactions are evaluated using standardized grading criteria that assess erythema, induration, and tenderness. The evaluation includes both objective measurements and subject- reported symptoms, with photography of injection sites when appropriate to document any significant reactions. Systemic reactions are monitored through a combination of clinical assessments and laboratory evaluations, including complete blood counts, comprehensive metabolic panels, and markers of inflammation.

[0214] Particular attention is paid to potential immune-mediated adverse events, given the sophisticated nature of these engineered immunogens. This includes monitoring for the development of autoantibodies and evaluation of any symptoms that might suggest immune complex formation or other immunological phenomena. The safety monitoring plan incorporates stopping rules based on predefined criteria for both individual participants and the overall study, ensuring appropriate protection of study participants while enabling collection of valuable safety data.

[0215] Immunogenicity Testing Methods43DM2\301045646.1PATENT Docket No. Y7969-99073

[0216] The evaluation of immune responses employs a hierarchical testing strategy that examines multiple aspects of the induced response. Serum antibody responses are initially assessed using enzyme-linked immunosorbent assays (ELISA) with both the immunogen and related proteins to evaluate binding specificity and cross-reactivity. The ELISA methodology incorporates careful attention to blocking conditions and wash steps to minimize background while maintaining sensitivity. Standard curves using well-characterized monoclonal antibodies enable quantitative comparison of responses between subjects and across timepoints.

[0217] Flow cytometric analysis of cellular responses requires careful attention to sample processing and storage conditions to maintain cell viability and function. Peripheral blood mononuclear cells are isolated using density gradient centrifugation within 4 hours of collection, with processed cells either analyzed immediately or cryopreserved in liquid nitrogen using a controlled-rate freezing protocol. The staining panels for flow cytometric analysis incorporate multiple markers to identify relevant B cell populations, with particular attention to memory B cells and plasmablasts that may indicate ongoing immune responses.

[0218] Data Analysis and Statistical Considerations

[0219] The analysis of immunological data requires sophisticated statistical approaches that account for the complexity and variability inherent in immune responses. Primary immunogenicity endpoints focus on the induction of apex epitope-specific B cells, with success criteria defined based on both the frequency and phenotypic characteristics of these cells. Statistical analysis employs mixed-effects models to account for repeated measurements within subjects, with appropriate corrections for multiple comparisons when examining responses across multiple timepoints or cell populations.

[0220] Sequence analysis of B cell receptors requires specialized bioinformatic approaches developed specifically for immunoglobulin repertoire analysis. Raw sequencing data undergoes quality filtering and preprocessing using established pipelines, with particular attention to the accurate identification of complementarity determining regions and framework regions. The determination of clonal relationships employs algorithms that consider both nucleotide sequences and amino acid translations, with parameters optimized for the identification of apex-specific antibody lineages. Somatic hypermutation analysis tracks the accumulation of mutations over time, providing insights into the maturation of the antibody response.

[0221] Alternative Formulations and Delivery Systems44DM2\301045646.1PATENT Docket No. Y7969-99073

[0222] Beyond the standard protein and mRNA formulations, the immunogens of the present invention may be delivered through alternative systems designed to enhance immune responses or facilitate administration. Nanoparticle formulations may incorporate the modified Env proteins in defined orientations and densities, with particle size and surface chemistry optimized for interaction with antigen-presenting cells. The nanoparticle composition may include biodegradable polymers, such as poly(lactic-co-glycolic acid) (PLGA), or self-assembling protein scaffolds that provide controlled release of the immunogen.

[0223] For mRNA delivery, alternative lipid compositions may be employed to modify tissue distribution or cellular uptake patterns. The lipid nanoparticle formulation may be modified through the incorporation of targeting ligands or surface modifications that enhance stability or cellular internalization. The ratio of various lipid components can be adjusted to optimize the balance between stability, cellular uptake, and endosomal escape, with the specific composition determined through systematic evaluation of particle characteristics and functional outcomes.

[0224] Scale-up Considerations and Manufacturing Process Development

[0225] The transition from research-scale to clinical-scale manufacturing requires careful attention to process scalability and reproducibility. The upstream process for protein production typically employs stirred-tank bioreactors with working volumes of 50-2000L. Process development includes optimization of key parameters such as cell density, nutrient feed strategy, and harvest timing to maximize protein yield while maintaining quality attributes. The production cell line undergoes extensive characterization, including evaluation of genetic stability and demonstration of freedom from adventitious agents.

[0226] Downstream processing must accommodate increased production volumes while maintaining product quality. Chromatographic steps employ larger columns with appropriate flow rates and loading conditions determined through small-scale studies and confirmed during process validation. The purification process incorporates viral clearance steps as required for clinical materials, with viral clearance studies conducted using relevant model viruses. In-process controls monitor quality attributes throughout the manufacturing process, with specifications established based on process capability and product requirements.

[0227] Product Specifications and Release Criteria

[0228] The establishment of comprehensive product specifications ensures consistent quality of the final immunogen preparations. For protein-based immunogens, specifications include45DM2\301045646.1PATENT Docket No. Y7969-99073 quantitative measures of purity, potency, and molecular characteristics. Purity assessments employ multiple orthogonal methods, including size exclusion chromatography to evaluate aggregate content, ion exchange chromatography to assess charge variants, and specific assays for host cell protein and DNA content. Potency testing incorporates both binding assays using reference antibodies and functional assays that evaluate the ability to induce specific B cell responses in standardized in vitro systems.

[0229] Specific acceptance criteria are established for each quality attribute based on manufacturing experience and correlation with biological function. Protein concentration specifications typically allow for a narrow range around the target value, with measurements performed using validated UV spectrophotometric methods. Trimer content must exceed a defined threshold, typically 90% as assessed by analytical ultracentrifugation or other appropriate methods. Endotoxin levels must remain below specified limits, with testing performed using validated kinetic chromogenic methods. Sterility testing employs both rapid methods for immediate release and traditional culture-based methods for definitive confirmation.

[0230] Troubleshooting Guidelines and Process Controls

[0231] The development and implementation of robust troubleshooting procedures ensures rapid identification and resolution of manufacturing or quality issues. Each process parameter is monitored through in-process controls with established action limits and investigation triggers. For protein expression, monitoring includes daily assessment of cell viability, metabolite levels, and protein production. Deviations from expected patterns trigger specific investigation pathways, with predetermined corrective actions based on the nature and severity of the deviation.

[0232] Analytical method troubleshooting follows systematic approaches designed to identify and correct sources of variability or error. Method-specific troubleshooting guides include decision trees that consider common failure modes and their resolution. For complex analytical methods such as flow cytometry or mass spectrometry, system suitability tests provide early indication of potential issues, enabling preventive maintenance or correction before impact on product testing. Documentation of troubleshooting activities includes root cause analysis and effectiveness verification of corrective actions.

[0233] Quality Control Measures for Flow Cytometry Analysis

[0234] Flow cytometry analysis requires specific quality control measures including:• Validation using fluorescence-minus-one (FMO) controls46DM2\301045646.1PATENT Docket No. Y7969-99073 • Standardized compensation using single-stained controls• Assessment of probe specificity using known positive and negative controls• Regular calibration of sorting parameters using standardized beads These measures ensure reliable identification and isolation of apex-specific B cells while minimizing false positives.

[0235] Future Development Considerations

[0236] The continuing development of HIV immunogens anticipates potential modifications and improvements based on emerging scientific understanding and clinical experience. Future iterations may incorporate additional modifications to enhance immunogenicity or broaden the spectrum of induced responses. These modifications may include alterations to glycosylation patterns, incorporation of additional stabilizing mutations, or development of heterologous primeboost strategies combining different immunogen variants.

[0237] Advanced delivery systems under consideration include controlled release formulations designed to optimize the kinetics of antigen presentation. These systems may employ biodegradable matrices or stimuli-responsive materials that provide sustained or triggered release of the immunogen. Alternative administration routes, such as intranasal or transdermal delivery, may be explored to enhance mucosal immunity or facilitate administration in resource-limited settings.

[0238] Regulatory Pathway and Requirements

[0239] The development pathway for HIV immunogens requires careful navigation of regulatory requirements across multiple jurisdictions. Initial regulatory submissions include detailed documentation of the manufacturing process, analytical methods, and preclinical safety studies. The chemistry, manufacturing, and controls (CMC) section requires particular attention to quality attributes and their control strategies. This includes comprehensive analysis of impurities, with specific attention to process-related impurities such as host cell proteins and potential aggregates, as well as product-related impurities including truncated forms or modified variants of the immunogen.

[0240] Preclinical safety assessment follows regulatory guidance specific to preventive HIV vaccines, incorporating both standard toxicology studies and specialized immunological evaluations. Safety studies examine both local and systemic effects, with particular attention to potential immune-mediated adverse effects. The toxicology program includes evaluation of repeat-47DM2\301045646.1PATENT Docket No. Y7969-99073 dose effects, examining multiple timepoints to assess both acute reactions and potential delayed effects. Additional studies may be required based on specific regulatory agency requirements or unique aspects of the immunogen design.

[0241] Clinical Development Strategy

[0242] The clinical development program typically proceeds through multiple phases, beginning with first-in-human studies focused on safety and initial immunogenicity. Phase I studies employ careful dose escalation with extensive safety monitoring, including evaluation of both local and systemic reactions. Immunogenicity assessment in early clinical studies focuses on demonstration of proof-of-concept, confirming the ability to induce the desired B cell responses in humans.

[0243] Later stage development incorporates larger studies designed to optimize dosing regimens and evaluate different immunization strategies. These studies may examine factors such as dose spacing, route of administration, and combination with other immunogens in heterologous prime-boost regimens. The clinical program includes development of appropriate assays for monitoring immune responses, with validation of these assays according to regulatory requirements for clinical trial use.

[0244] In an aspect, the immunogens and methods described herein represent a comprehensive approach to inducing broadly neutralizing antibodies against HIV, particularly targeting the apex epitope of the HIV envelope protein. In an aspect, the engineered immunogens incorporate multiple strategic modifications designed to enhance binding to bnAb precursors while maintaining proper protein folding and stability. In an embodiment, these modifications include carefully selected mutations in the V1 / V2 region, optimization of glycosylation patterns, and structural elements that promote proper trimerization.

[0245] Diagnositc and Therapeutic Methods

[0246] The immunogens and antibodies of the present invention provide significant utility in both diagnostic and therapeutic applications. In an aspect, for therapeutic treatment of HIV infection, the antibodies may be administered through various routes, with intravenous, subcutaneous, or intramuscular delivery being preferred. Therapeutic dosing typically ranges from 1-50 mg / kg, with 2-20 mg / kg being particularly effective. In an aspect, the dosing interval can be adjusted based on antibody half-life and clinical response, with typical intervals ranging from 2-8 weeks.48DM2\301045646.1PATENT Docket No. Y7969-99073

[0247] In diagnostic applications, the antibodies demonstrate high sensitivity in detecting HIV Env across various biological sample types. These samples may include blood, serum, plasma, tissue biopsies, and mucosal swabs. The detection methodology can employ various labeling strategies. Fluorescent molecules provide sensitive detection with minimal background, while enzymatic labels offer amplification capabilities for enhanced sensitivity. In an aspect, the detection limit typically reaches 1-100 pg / mL of HIV Env protein, enabling early detection of viral infection.

[0248] Detection Methods and Kits

[0249] The immunogens described herein enable identification of subjects possessing B cell precursors capable of developing into broadly neutralizing antibodies. Detection strategies employ immunogens labeled with detectable markers appropriate for the intended application. Fluorescent labeling provides direct visualization capabilities, while enzymatic labels enable signal amplification for enhanced sensitivity. Biotin-streptavidin systems offer flexibility in detection strategies through their modular nature. Metal nanoparticles provide stability and multiplex detection capabilities, while radioactive labels enable highly sensitive detection when appropriate containment measures are available.

[0250] The detection of B cells binding to labeled immunogens can be accomplished through multiple complementary approaches. Fluorescence-based detection enables sensitive identification of binding events, while magnetic separation allows efficient isolation of specific cell populations. Affinity chromatography provides a scalable approach for isolating specific B cells, and various imaging techniques enable detailed characterization of binding events.

[0251] For specific identification of apex epitope-recognizing B cells, knockout variants lacking the apex epitope serve as controls. These variants, generated by introducing mutations R169E and K171E, enable differential identification of B cells recognizing the apex epitope versus other epitopes on the immunogen surface. This approach ensures isolation of B cells with the desired epitope specificity.

[0252] Comprehensive kits for detecting apex epitope-specific B cells incorporate the necessary components for reliable detection and isolation. The wild-type immunogen and knockout variant, each bearing distinct labels, enable differential detection. Supporting reagents including blocking solutions and wash buffers ensure specific binding events. Detection reagents49DM2\301045646.1PATENT Docket No. Y7969-99073 and cell isolation materials enable identification and recovery of desired cell populations. Detailed protocols guide users through the complex procedures while minimizing technical variation.

[0253] Nucleic-Acid Based Methods

[0254] Nucleic acid-based immunization approaches expand the available delivery options for the immunogens. The mRNA encoding the immunogen can be effectively delivered through various carrier systems, with lipid nanoparticles showing particular promise. Alternative approaches including polymeric nanoparticles, electroporation, and direct injection provide flexibility in delivery strategy selection.

[0255] The mRNA doses typically range from 10-100 pg per administration, with expression of the immunogen detectable within 6-48 hours. Peak expression levels occur at 24-36 hours postadministration. Multiple administrations may be performed at 3-8 week intervals to enhance and maintain the immune response.

[0256] Lipid nanoparticle formulations for mRNA delivery comprise carefully optimized combinations of ionizable lipids, helper lipids, cholesterol, and PEG-modified lipids. These formulations typically produce particles ranging from 80-200 nm with minimal size distribution variation. The encapsulation efficiency consistently exceeds 80%, ensuring efficient delivery of the mRNA cargo.

[0257] Immunization Strategies

[0258] The invention enables flexible immunization strategies through various prime-boost approaches. Initial immunization with mRNA-LNP followed by protein boosting can effectively induce robust immune responses. Alternatively, protein prime followed by mRNA-LNP boost or alternating administrations of different formulations provide additional strategic options. The selection of specific strategies depends on multiple factors including the magnitude and quality of B cell responses, duration of immunity, and practical implementation considerations.

[0259] Comprehensive immune monitoring strategies enable evaluation of vaccination effectiveness. This includes quantification of serum antibody titers, detailed B cell phenotyping, functional antibody assays, and structural studies of elicited antibodies. This thorough characterization ensures proper evaluation of immune response quality and guides optimization of vaccination strategies.

[0260] The development process encompasses multiple complementary approaches, from initial protein engineering through preclinical evaluation and preparation for clinical studies. The50DM2\301045646.1PATENT Docket No. Y7969-99073 manufacturing processes described provide a robust platform for consistent production of clinical-grade material, with appropriate controls and specifications to ensure product quality. The analytical methods and characterization techniques enable comprehensive evaluation of quality attributes and demonstration of lot-to-lot consistency.

[0261] The immunological evaluation strategies described herein provide detailed understanding of the induced immune responses, with particular attention to the development of apex-specific antibodies with desired characteristics such as extended HCDR3 regions and specific sequence motifs. The combination of cellular and molecular analyses enables tracking of both the phenotypic and genetic characteristics of the induced response, providing insights into the effectiveness of the immunization strategy.

[0262] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0263] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.ExamplesExample 1: Induction of HIV broadly neutralizing antibody precursors to the Apex epitope in nonhuman primates

[0264] A primary goal for the HIV vaccine design field is to develop a vaccine that induces broadly neutralizing antibodies (bnAbs) that can protect against diverse HIV strains 1-4). One of the leading strategies proposed to achieve that goal, germline-targeting vaccine design, requires a priming immunogen to induce rare bnAb-precursor B cells that possess the genetic features needed to develop into bnAbs, followed by a series of heterologous boost immunogens to guide maturation to bnAb development (5-28). The IAVI G001 clinical trial demonstrated that a germline-targeting immunogen induced precursors for the VRC01 class of bnAbs specific for the CD4 binding site of HIV Env, in 97% of vaccine recipients (19, 20). However, a vaccine that elicits VRC01-class bnAbs in humans has not yet been reported, as developing a sequential boosting regimen to guide bnAb-precursor B cells to acquire the mutations needed to become bnAbs remains a challenging and complex task (15, 29-31). Furthermore, an optimal vaccine capable of inducing bnAbs to all51DM2\301045646.1PATENT Docket No. Y7969-99073 or nearly all circulating HIV strains will require induction of multiple different classes of bnAbs specific for different HIV Env epitopic regions.

[0265] The development of ApexGT6 was guided by the observation that Apex broadly neutralizing antibodies (bnAbs) require fewer somatic hypermutations but longer HCDR3s compared to other HIV bnAbs. BnAbs specific for the Apex region of Env include some of the most potent bnAbs known and typically have less somatic hypermutation (SHM) than bnAbs to other Env epitopes (32, 33), making them desirable targets for vaccination as in theory they might be elicited by fewer sequential heterologous boosting steps than other bnAb classes. The primary hurdle to eliciting Apex bnAbs is believed to be the low frequency of bnAb-precursor B cells possessing a long (> 24 amino acid [aa]) and acidic HCDR3 that can penetrate the glycan shield and bind to the positively charged surface within the Apex epitopic region (18, 33-36).

[0266] Among the long-HCDR3 Apex bnAbs, PCT64 and PG9 were identified as having the highest and second-highest precursor frequencies in the human immunoglobulin repertoire (18). Furthermore, a germline-targeting Env trimer, ApexGT5, was shown to have affinity for the inferred precursors of both PCT64 and PG9 (18) and to be capable of priming PCT64-like responses in a mouse model (17).

[0267] To engineer an improved immunogen, structure-guided directed evolution was performed using mammalian cell-surface display. Two libraries were constructed and screened. The first library employed random mutagenesis targeting the VI V2 region using error-prone PCR. The second library used combinatorial saturation mutagenesis targeting specific residues identified within the PCT64 antigen-antibody binding interface. Selection was performed using the least mutated common ancestor of PCT64 (PCT64 LMCA) as a positive probe and the non-neutralizing antibody B6 as a negative probe. This dual selection strategy enabled identification of mutations that enhanced affinity to germline antibodies while maintaining proper trimerization.

[0268] The resulting immunogen, ApexGT6, was developed in two forms to enable different delivery strategies. The first form, designated ApexGT6 congly, was designed as a soluble protein trimer containing three key mutations compared to the previous ApexGT5 design (Table 1). To minimize off-target responses to glycan holes outside the apex region, N241 and N289 glycosylation sites were introduced through congly mutations P240Q, S241N, and P291S. The second form, designated ApexGT6 L14, was engineered as a cleavage-independent version capable of being expressed as a membrane-bound trimer (gp151) to facilitate mRNA-LNP52DM2\301045646.1PATENT Docket No. Y7969-99073 immunization. This was achieved by introducing a 14-amino-acid linker (linkl4, L14, “SHSGSGGSGSGGHA (SEQ ID NO: 11)”) in place of the furin cleavage site (REKR, 508-511, HXB2 numbering), eliminating the need for furin cleavage. Both the congly and L14 mutations are applicable to MD39, ApexGT5, and ApexGT6.Table 1. Mutations in ApexGT variants compared to BG505 MD39Position MD39 ApexGT5 ApexGT6 Functional Role (HXB2)P76 P P S Stabilizes V1V2 apex conformationS128 T T G Enhanced binding to germline precursors I161 M A A Improves recognition by PCT64-class antibodies G167 D N N Improves recognition by PCT64-class antibodies V169 K R R Improves recognition by PCT64-class antibodies L179- LDVVQINENQG LDIVPMVD LDIVPMVD Enhanced binding to Y191 NRSNNSNKEY LWTNY LWTNY germline precursors (SEQ ID NO: 12) (SEQ ID NO: (SEQ ID13) NO: 13)N195 N N S Stabilizes V1V2 apexconformation

[0269] Comprehensive characterization of ApexGT6 variants was performed using multiple complementary techniques. Surface plasmon resonance (SPR) analysis demonstrated enhanced affinity for both PCT64 and PG9 precursors that had been identified through next-generation sequencing of HIV seronegative individuals. The improved binding characteristics are shown in FIG. 1C, with significant enhancements in affinity compared to previous designs.

[0270] Antigenic profiling by enzyme-linked immunosorbent assay (ELISA) confirmed that both ApexGT6 congly and L14 exhibited the antigenic profile of well-folded Env trimers. The proteins showed strong binding to trimer-specific antibodies while maintaining minimal reactivity with non-neutralizing epitopes, as demonstrated in FIG. 8B. Thermal stability was assessed by differential scanning calorimetry, which revealed excellent stability with melting temperatures of53DM2\301045646.1PATENT Docket No. Y7969-99073 71.48°C for ApexGT6 and 70.91°C for ApexGT6 congly, comparable to the previous ApexGT5 designs.

[0271] The glycosylation profile of the immunogens was analyzed using the DeGlyPHER method, which enabled comprehensive characterization of all N-linked glycosylation sites. Both ApexGT6 variants showed good occupancy at the highly conserved glycosylation sites within the apex region, as detailed in FIG 8D. Proper trimerization and oligomeric state were confirmed through size exclusion chromatography coupled with multi-angle light scattering analysis.

[0272] The quality of ApexGT6 preparations underwent comprehensive analysis using multiple orthogonal methods. Analytical ultracentrifugation demonstrated consistent trimer content exceeding 90%, with proper molecular weight of 440 ± 20 kDa and a sedimentation coefficient of 13.5 ± 0.5 S. Size exclusion chromatography analysis revealed a single symmetric peak with retention time consistent with trimeric species and aggregate content below 5%. Thermal stability studies showed melting temperatures of 71.48°C for ApexGT6 and 70.91°C for ApexGT6 congly, with single thermal transitions indicating cooperative unfolding of the trimeric structure.Example 2: Validation of Non-Human Primates as Pre-clinical Models for Apex Vaccine Development

[0273] Prior to immunization studies, extensive analysis was performed to evaluate rhesus macaques (RMs) as a relevant pre-clinical model for Apex vaccine development. RMs have a relatively human-like naive B cell repertoire and the ability to produce long HCDR3s (37), and a RM infected with a simian-human immunodeficiency virus (SHIV) produced an Apex epitopespecific bnAb, RHA1, similar to the PCT64 human bnAb (35). The inventors sought to evaluate whether Apex bnAb-related precursors could be induced by vaccination in outbred primates that can produce polyclonal long-HCDR3 responses, including not only bnAb precursors but also long-HCDR3 competitors absent from mouse models. The inventors designed a slightly improved germline-targeting trimer, ApexGT6, with affinity for additional PCT64- and PG9-class precursors, and the inventors evaluated B cell responses to this trimer delivered as adjuvanted soluble protein or mRNA-encoded membrane-anchored protein in RMs. The analysis focused on comparing the genetic features and precursor frequencies between human and RM B cell repertoires that would be relevant for developing Apex bnAb responses.54DM2\301045646.1 PATENT Docket No. Y7969-99073

[0274] Initial genetic analysis revealed that RMs possess a germline D gene (DH3-41) with several alleles similar to the human DH3-3. To compare frequencies of PCT64-like heavy chain (HC) precursors utilizing these genes, bioinformatic searches were conducted in ultra-deep immunoglobulin HC sequencing datasets from both humans and RMs. The analysis included 14 human donors and 60 RMs. PCT64-like precursors were defined by three key criteria: (1) HCDR3 length > 24 amino acids (one amino acid shorter than PCT64); (2) HCDR3 encoded by DH3-41 or DH3-3 using the same reading frame as PCT64; and (3) D gene positioning approximately in the middle of the HCDR3, specifically at least 6 amino acids from the beginning and 12 amino acids from the end, based on the position in PCT64.

[0275] This analysis detected PCT64-like precursors in 57 of 60 RMs (95%) and all 14 human donors. Among RMs with detectable DH3 -41 -based PCT64-like HC precursors, the frequency was 171.8 per million, approximately 7.5-fold lower than the frequency of DH3-3-based PCT64-like precursors in humans (1,288 per million). These results suggested that vaccine priming of DH3-41 -based PCT64-like responses in RMs might be more challenging than human vaccine priming of DH3-3-based PCT64-like responses.

[0276] Further analysis revealed an important compensatory feature in the RM repertoire. RMs possess another germline D gene, DH3-15, which directly encodes a DDY motif that is present at the tip of the long HCDR3s of several important Apex bnAbs, including RHA1 (the only known Apex epitope-specific bnAb isolated from a RM), PCT64, and PGDM1401 (77) (FIG. IF). Structural studies of these bnAbs show that DDY includes a sulfated tyrosine (72) and makes key interactions with Env (18, 38) (FIG. 1G). The DDY in the human bnAbs most likely resulted from a V(D)J recombination event or was acquired during lineage development through somatic hypermutation (SHM) (73), unlike the DDY in RHA1 germline-encoded by DH3-15.

[0277] Using RHA1, PCT64, and PGDM1401 as guides, a more general class of Apex bnAb-related precursors based on the following criteria: (1) HCDR3 length > 24 aa; and (2) the DDY motif occurred approximately in the middle of the HCDR3, at least 7 aa from the beginning and 10 aa from the end, based on the position in PCT64 and RHA1 (FIG. 8E). These definitions allowed for precursors with exceptionally long HCDR3s possessing a DDY motif, regardless of germline gene usage, with diverse V-D and D-J junctions. Although humans lack a germline D gene that can directly encode DDY on the HCDR3 (FIG. 8F), DDY could occur in human HCDR3s through V-D or D-J junctions. Analysis revealed DDY-containing precursors in 59 of 60 RMs55DM2\301045646.1 PATENT Docket No. Y7969-99073 (98.3%) and all 14 humans (FIG. 1H). The frequency of such precursors was 7.2-fold higher in RMs (median 454.9 per million) than in humans (median 63.12 per million) (FIG. 1H), likely owing to the DDY-encoding DH3-15 gene in RMs for which humans have no counterpart. These results suggested that it might be easier to elicit Apex bnAb-related DDY-encoded responses in RMs than in humans.

[0278] Additional analysis of PG9-like precursors was performed using similar criteria but with modified requirements: (1) HCDR3 length > 29 aa (1 aa shorter than PG9); (2) HCDR3 encoded by DH3-41 or DH3-3 using the same reading frame as PG9; and (3) the start position of the D gene occurred approximately in the middle of the HCDR3, at least 15 aa from the beginning and 8 aa from the end, based on the position in PG9 (FIG. 8E). Such precursors were detected in 14 of 14 humans but only 6 of 60 RMs, and the median frequency among responders was 6.5-fold lower in RMs (median among responders, 4.8 per million) than in humans (median 31.6 per million) (FIG. 8G). These results suggested that eliciting PG9-like responses in RMs could be substantially more challenging than in humans.

[0279] The comprehensive analysis identified two distinct sets of Apex bnAb-related precursors in RMs with frequencies comparable to or exceeding those in humans: 1. PCT64-like precursors utilizing DH3-41, though at lower frequencies than human DH3-3-based precursors; 2. DDY-containing precursors at higher frequencies than in humans due to the DH3-15 gene. These findings supported the use of RMs as relevant preclinical models for evaluating ApexGT priming immunogens, while highlighting specific considerations for interpreting results, particularly regarding PG9-like responses. The data suggested that RMs could effectively model certain aspects of human responses while potentially offering advantages for studying DDY-dependent neutralization mechanisms.Example 3: Immunization Studies and Serum Response Analysis

[0280] ApexGT6 immunization studies were conducted in rhesus macaques using two distinct delivery platforms. ApexGT6 immunization studies were conducted in rhesus macaques using two distinct delivery platforms. The study design included four groups of six RMs each, with all immunizations performed at weeks 0 and 8. Immunizations were administered bilaterally in the deltoid area as follows:56DM2\301045646.1PATENT Docket No. Y7969-99073 Group 1 (ApexGT6 Protein):• Subcutaneous immunization• 50 pg of soluble ApexGT6 congly protein• 375 pg of saponin / MPLA nanoparticle (SMNP) adjuvant per animal flank Group 2 (ApexGT6 mRNA):• Intramuscular immunization• 50 pg of mRNA-LNP encoding membrane-bound ApexGT6 (ApexGT6 L14 gpl51)Control GroupsGroup 3:• Intramuscular immunization• 50 pg of soluble trimer BG505 MD39.3• 375 pg of SMNPGroup 4:• Intramuscular immunization• 50 pg of membrane-bound BG505 MD39.3 gp 151 mRNA-LNP

[0281] SAMPLE COLLECTION AND PROCESSING

[0282] Throughout the study, the following samples were collected at multiple timepoints (FIG. 2A):• Blood in NaCitrate CPT tubes for peripheral blood mononuclear cells (PBMCs) and plasma isolation• Serum via serum clot tubes• Lymph node fine needle aspirates (FNA) (45) of axillary lymph nodes (LNs)

[0283] Protein Immunization Group Results

[0284] After the second immunization, all 6 animals produced serum IgG binding to the Apex epitope of ApexGT6 (FIG. 2B and FIG. 9A). Negative-stain electron microscopy polyclonal epitope mapping (nsEMPEM) demonstrated Vl / V2-directed antibody responses present in all ApexGT6-immunized animals at week 10 (FIGs. 2C-D).

[0285] The importance of the germline-targeting design was validated through control experiments. RMs immunized with BG505 MD39, a soluble native-like Env trimer lacking57DM2\301045646.1PATENT Docket No. Y7969-99073 germline targeting mutations but based on the same HIV isolate as ApexGT6, failed to produce an Apex epitope-specific response (FIG. 9B). This confirmed that the ApexGT modifications were both necessary and sufficient for triggering Apex epitope-specific serum IgG responses.[002861 mRNA-LNP Immunization Control Group Results

[0287] The mRNA-LNP immunization group demonstrated strong serum responses to the Apex epitope (FIG. 4A), with approximately 50% of the ApexGT6-specific response directed to the Apex epitope (FIG. 11 A). Comparative analysis between immunization platforms revealed interesting differences: protein immunization resulted in higher overall titers to the immunogen (FIG. 11B), and the magnitude of the Apex epitope-specific response (FIG. 11C) and the percentage of serum IgG titers that were Apex epitope-specific (FIG. 4B) were both higher in the membrane-bound mRNA group.

[0288] This superior epitope focusing was achieved despite the soluble protein having two glycan holes filled while the mRNA immunogen did not, suggesting inherent advantages of the mRNA-delivered membrane-bound platform for eliciting Apex epitope-directed responses.

[0289] When attempting nsEMPEM analyses of serum-derived polyclonal Fabs from the mRNA immunization group mixed with soluble ApexGT6 trimer, the Fab-Env complexes were predominantly monomeric. This prevented obtaining sufficient trimer-liganded classes for 3D volume reconstruction. A similar phenomenon was observed in the membrane-bound native-like MD39 Env mRNA-immunized group, suggesting that antibodies elicited by membrane-bound trimers may interact with soluble trimers in ways that promote trimer degradation during sample preparation.

[0290] Detailed ELISA analysis was performed using the following protocol: 96-well plates were coated overnight at 4°C with 6x-His (SEQ ID NO: 18) Epitope Tag Rabbit Antibody (Genscript, Cat. No. A00174) at 2ug / mL in PBS. Plates were washed 3 times with PBS, 0.2% Tween (PBS-T), and blocked with 5% milk and 1%FBS in PBS-T for Ih. Subsequently, 2ug / mL of the purified His-tagged trimers was added for 2h in PBS, after which the plates were washed three times with PBS-T. Serial dilutions of antigenic profiling mAbs in 1% FBS PBS-T were added to the plates for 1 h, after which the plates were washed again three times with PBS-T before the addition of anti-human conjugated peroxidase at 1:5000 for 1 h. After four final washes, binding was detected by the addition of TMB substrate and measured by absorbance at 450 and 570 nm. Background subtraction was performed by subtracting the 570 nm58DM2\301045646.1PATENT Docket No. Y7969-99073 value from the corresponding 450 nm value. Data were subsequently analyzed in Prism (Prism vl0.2; GraphPad Software).

[0291] Binding specificity was assessed by comparing responses to: intact immunogen (labeled as “live”) and apex-binding knockout variant (labeled as “KO”). Clear epitope-specific responses were observed following ApexGT6 immunization but not with the control MD39 immunogen.

[0292] Polyclonal antibody responses were further characterized using negative-stain EM:1. IgG was purified from heat-inactivated plasma samples2. Purified IgG was digested into Fab fragments3. Fab-trimer complexes were formed and purified4. Samples were analyzed using standardized EM protocols

[0293] This analysis revealed distinct antibody targeting patterns between ApexGT6 and MD39 immunized animals, with ApexGT6 immunization specifically inducing recognition of the apex epitope region.

[0294] The results demonstrated successful induction of Apex epitope-specific responses through both immunization platforms, with each showing distinct advantages. The mRNA platform showed superior epitope focusing, while the protein platform generated higher overall titers. These findings provide important insights for optimizing vaccination strategies targeting the HIV Apex epitope.Example 4: Analysis of Germinal Center and Memory B Cell Responses

[0295] Successful priming by a germline-targeting immunogen must produce a strong germinal center (GC) and / or memory B cell response derived from bnAb-precursor B cells (MBC). To investigate whether ApexGT6 could indeed prime bnAb-precursor B cells, the inventors performed B cell sorting and receptor sequencing on ApexGT6-binding B cells from postimmunization LN and PBMC samples.

[0296] Cell populations were defined as LN B cells (CD20+CD38‘), mostly GC B cells (BGC) referred to as such, and PBMC memory B cells (Bmem), defined as CD20+IgD‘.

[0297] Specificity for ApexGT6 was determined using ApexGT6 probes conjugated to two different fluorophores (ApexGT++) (FIGs. 9C-D). Specificity for the Apex epitope was determined by differential binding to ApexGT6 and ApexGT6. KO, a variant of ApexGT6 with two mutations59DM2\301045646.1PATENT Docket No. Y7969-99073 in the Apex region (R169E and K171E in HxB2 numbering) that essentially abrogates binding by long-HCDR3 Apex bnAbs and related precursors (77). Apex epitope-specific B cells were defined as ApexGT6++ApexGT6. K0‘.[002981 FLOW CYTOMETRY METHODOLOGY

[0299] The BGC and Bmem cellular dynamics were measured by flow cytometry. The protocol included:1. Sample Preparation:• LN and PBMC samples were thawed and recovered in fully supplemented RPMI • Live cells were counted and stained sequentially with probes and antibodies2. Staining Protocol:• Sequential staining with ApexGT6. KO and ApexGT6 probes (20 minutes each at 4°C) • Additional staining with antibody master mix• Anti-human hashtag antibodies (2.5 pg per 5 million cells)• PBS with 2% FBS used as FACS buffer3. Quality Control Measures:• Probe specificity validated using control antibodies• Multiple fluorophore combinations to confirm binding patterns• Technical replicates for consistency• Post-sort purity verification (>95%)

[0300] ANALYSIS OF GERMINAL CENTER RESPONSES IN PROTEIN IMMUNIZATION GROUP

[0301] In the protein immunization group, flow cytometry analysis revealed significant expansion of ApexGT6++BGC cells after immunization

[0302] In the protein immunization group, flow cytometry analysis revealed a significant expansion of ApexGT6++BGC cells after immunization, with median frequency increasing 5.8-fold from week 2 to week 12 (FIG. 2E). Importantly, the fraction of Apex epitope-specific BGC cells among ApexGT6++BGC cells remained consistently high throughout this interval, with median values ranging from 57% to 79% (FIG. 2F).

[0303] The memory B cell compartment showed even more dramatic expansion, with frequencies of ApexGT6++Bmem cells increasing approximately 34-fold from 0.017% at week 8 to60DM2\301045646.1PATENT Docket No. Y7969-99073 0.572% at week 10 following the second immunization (FIG. 2G). While these frequencies declined approximately 8-fold to 0.069% by week 17, the fraction of Apex epitope-specific Bmem cells among ApexGT6++Bmem cells showed continuous increase during this period, reaching a median value of approximately 69% by week 17 (FIG. 2H).

[0304] For the mRNA-LNP immunization group, robust memory B cell responses were observed in the peripheral blood, with significant increases in ApexGT6++Bmem cell frequencies following the second immunization (FIG. 4C). The fraction of Apex epitope-specific Bmem cells among ApexGT6++Bmem cells remained consistently high, with median values ranging from 60% to 80% (FIG. 4D). While minimal ApexGT6-specific cell responses were detected in the sampled lymph nodes from this group, the strong Bme responses in blood suggested that the intramuscularly administered mRNA-LNP immunizations likely drained to lymph nodes that were not accessible for FNA sampling.

[0305] BCR sequencing analysis yielded 8,168 ApexGT6-negative and 2,376 ApexGT6++paired (heavy and light chain) BCR sequences from 20 FNA samples and 678 ApexGT6++BCR sequences from 9 PBMC samples in the protein immunization group (FIG. 10A and Table 2).Table 2. BCR Sequence Analysis from Protein Immunization GroupSample Total BCRs ApexGT6++Epitope- Long HCDR3 Apex Type Analyzed BCRs Specific BCRs (>24aa) bnAb-like FNA GC 8,168 2,376 1,707 331 312 PBMC 678 628 33 21 21Memory

[0306] For the mRNA-LNP group, 1,401 paired heavy and light chain BCR sequences were obtained from PBMCs of the six immunized animals (Table 3).Table 3. BCR Sequence Analysis from mRNA-LNP Immunization GroupAnimal Total ApexGT6++Epitope- Long HCDR3 Apex bnAb- ID BCRs BCRs Specific (>24aa) like RAul8 48 48 4 4 4 RGul8 43 43 10 10 10RJr18 399 399 118 99 99 RPbl9 83 83 14 11 11 RWjl8 760 543 97 92 92RYul8 68 68 2 2 261DM2\301045646.1PATENT Docket No. Y7969-99073Total 1,401 1,184 245 218 218

[0307] Full-length V(D)J reads were assembled with CellRanger v.3.0, utilizing a custom Rhesus Macaque germline VDJ library (46, 47, 86) and adjusting the constants.py file for a maximum CDR3 length of 110 nucleotides. Gene expression counts were obtained from gene expression libraries using CellRanger v.6, aligning to the Ensemble MmullO genome and incorporating mitochondrial genes from Mmul9. Sequencing data were demultiplexed using the MULTIseqDemux command in Seurat v.4. For B cells with kappa and lambda light-chain contigs, lambda was assigned when both were present. The VDJ sequences were then parsed into an AIRR format using the Change-O pipeline from the Immcantation portal for comprehensive analysis.

[0308] Analysis of these sequences revealed a striking enrichment of long HCDR3s with length > 22 amino acids in ApexGT6++and Apex epitope-specific BCRs, but not in BCRs that did not bind to ApexGT6 (FIG. 3A and FIG. 10B). This enrichment pattern was observed in both immunization groups and across multiple timepoints, demonstrating the consistent ability of ApexGT6 to engage and activate B cells bearing long HCDR3s characteristic of Apex bnAbs.Example 5: Characterization of Apex bnAb-like Precursor Responses

[0309] Detailed analysis focused on BCRs with HCDR3 lengths equal to or longer than 24 amino acids, as this represents the minimum length typically required for Apex bnAbs to penetrate the glycan shield (18, 38).

[0310] In the protein immunization group, 331 Apex epitope-specific paired BCRs with long HCDR3s (> 24 aa) were identified in five out of six animals. VDJ alignment analysis revealed that ApexGT6++and Apex epitope-specific BCRs with long HCDR3s were nearly entirely (99%) composed of sequences utilizing IGHD3-15. In contrast, in a control dataset of naive BCR sequences from 60 RMs (24), less than 22% of BCRs with long HCDR3s used IGHD3-15 (FIG.3B and FIG. 10C). In addition, none of the Apex epitope-specific GC BCRs met the DH3-41 precursor criteria described above. However, the inventors observed that more than 94% of Apex epitope-specific GC BCRs with long HCDR3s elicited by ApexGT6 immunization met the Apex bnAb-like precursor criteria described earlier (FIG. 10E, “RM DDY”), suggesting that the majority of these sequences were Apex bnAb-like precursors.62DM2\301045646.1PATENT Docket No. Y7969-99073

[0311] While none of the Apex epitope-specific GC BCRs met the previously defined DH3-41 precursor criteria, more than 94% of Apex epitope-specific GC BCRs with long HCDR3s elicited by ApexGT6 immunization met the Apex bnAb-like precursor criteria defined in FIG. 8E. The frequency of these Apex bnAb-like precursors among ApexGT6++GC BCRs increased over time (FIG. 3C), reaching a median frequency of 5% across responding animals.

[0312] In PBMC samples from three of the six protein-immunized animals, 21 Apex bnAb-like precursors were identified at weeks 10 and 17. Notably, in one animal (RPzl8), six different Apex bnAb-like precursor lineages were detected in PBMCs despite none being found in FNA samples (FIGs. 3C-D), suggesting potential underestimation of response breadth due to sampling limitations. Among animals with detectable Apex bnAb-like precursors in PBMCs, the median frequency among ApexGT6++memory BCRs was 3.8% (FIG. 3D).

[0313] The mRNA-LNP immunization group showed similar success in eliciting Apex bnAb-like precursors. Analysis of Apex epitope-specific BCRs revealed an enrichment of long HCDR3s (FIG. 4E and FIG. 11E), with 96% of these sequences containing IGHD3-15, and more than 88% meeting the Apex bnAb-like precursor criteria (FIG. 4F).

[0314] In total, 333 paired Apex bnAb-like precursor BCRs were identified from the protein immunization group, clustering into 30 unique clonal lineages (FIG. 3E). From the mRNA-LNP group, 218 paired Apex bnAb-like precursor memory BCRs were identified, clustering into 80 unique lineages across all six animals (FIG. 4G). These lineages exhibited diverse HCDR3 lengths and unique junctional sequences, indicating they arose from distinct precursors.

[0315] Comparative analysis between the two immunization platforms revealed that while the protein group showed higher frequency of ApexGT6++Bman cells among total Bmem cells (FIG.41), the mRNA-LNP group demonstrated a higher percentage of Apex bnAb-like precursors among ApexGT6++memory BCRs (FIG. 41). Notably, the mRNA-LNP immunized animals produced a higher frequency of Apex bnAb-like precursors with very long HCDR3s (length > 26 aa) compared to protein immunization (FIG. 4K and FIG. 11F). This feature could be particularly important as human Apex bnAbs with longer HCDR3s typically exhibit greater breadth and potency (FIG. 11G-H).

[0316] Clonal lineage determination employed a systematic approach combining both sequence- and structure-based criteria. Initial clustering used a distance metric incorporating V-gene identity, junction length, and amino acid similarity within the HCDR3. Putative lineage63DM2\301045646.1PATENT Docket No. Y7969-99073 assignments underwent additional validation through phylogenetic analysis and evaluation of shared somatic mutations. The identification of convergent features across independent lineages provided strong evidence for selective pressure driving similar structural solutions for apex recognition.

[0317] Key differences between platforms were observed: adjuvanted-protein immunization induced a stronger ApexGT6++Bmem response, whereas the mRNA-encoded membrane-bound platform induced an ApexGT6++Bmem response with superior focusing on the Apex epitope (which was consistent with the serum results). The balance of these factors resulted in protein and mRNA-LNP inducing similar overall frequencies of Apex bnAb-like precursors among Bmem cells.

[0318] These findings demonstrate successful induction of Apex bnAb-like precursors through both immunization platforms, with each showing distinct advantages. The complementary nature of these advantages suggests potential benefit in combining both approaches in future vaccination strategies.Example 6: Comparison of Protein and mRNA-LNP Immunization Platforms

[0319] To test if mRNA immunization strategies could be used to prime Apex epitope-specific responses, a comparative study was conducted between traditional protein immunization and mRNA delivery. Six RMs were immunized with 50 pg of mRNA-LNP encoding cleavageindependent membrane-bound ApexGT6 (ApexGT6 L14 gpl 51), bilaterally and intramuscularly (IM) in the deltoid. The experimental timeline and sample collections were identical to the adjuvanted-protein group (FIG. 2A).

[0320] After two immunizations with mRNA-LNP, all six animals produced serum IgG that bound to the Apex epitope (FIG. 4A). Approximately 50% of the ApexGT6-specific response was directed to the Apex epitope (FIG. 11 A). In a control experiment, RMs were immunized with mRNA encoding a native-like Env trimer (BG5O5 MD39) based on the same HIV isolate as ApexGT6 and also designed as a cleavage-independent membrane-bound trimer. BG505 MD39 mRNA induced an Apex epitope-specific response that was significantly weaker than the ApexGT6 mRNA response (FIG. 11 A). When comparing serum IgG titers between the ApexGT6 protein and mRNA groups, protein immunization resulted in higher titers to the immunogen (FIG.1 IB). However, the magnitude of the Apex epitope-specific response (FIG. 11C) and the percentage of serum IgG titers that were Apex epitope-specific (FIG. 4B) were both higher in the64DM2\301045646.1PATENT Docket No. Y7969-99073 membrane-bound mRNA group. This was true even though the soluble protein had two glycan holes filled, while the mRNA immunogen did not, suggesting that the mRNA-delivered membrane-bound platform may be more effective than adjuvanted soluble protein in eliciting an Apex epitope-directed response.

[0321] Although the ELISA showed a strong serum response to the Apex epitope in the mRNA group, negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) analyses revealed that, after mixing serum-derived polyclonal Fabs from the mRNA immunization group with soluble ApexGT6 trimer, the Fab-Env complexes were predominantly monomeric. As a result, it was not possible to obtain enough trimer-liganded classes to reconstruct 3D volumes. A similar issue was also observed in the membrane-bound native-like MD39 Env mRNA-immunized group, suggesting that the antibodies in the post membrane-bound trimer immunized plasma induce degradation of soluble trimers during sample preparation of nsEMPEM.

[0322] Next, the germinal center and memory cellular dynamics were measured following ApexGT6 mRNA-LNP immunization via flow cytometry. In PBMCs, the frequencies of ApexGT6++Bmem cells significantly increased after the second immunization (FIG. 4C). The fraction of Apex epitope-specific Bmem cells among ApexGT6++Bmem cells remained consistently high during that period, with median values ranging from 60% to 80% (FIG. 4D). In contrast, in LN samples, minimal ApexGT6-specific cell responses were observed. Given the robust Bmem response seen in the blood, the inventors concluded that IM mRNA-LNP immunizations predominantly drained to an inaccessible LN, limiting the information obtained from the axillary LN FNAs. Nevertheless, two immunizations of mRNA encoding membrane-bound ApexGT6 induced strong Apex epitope-specific Bmem cell responses.

[0323] The inventors obtained 1,401 paired heavy and light chain BCR sequences from the PBMCs of the 6 animals immunized with mRNA-LNPs (Table 3). The inventors observed an enrichment of long HCDR3s (> 24 aa) in Apex epitope-specific BCRs (FIG. 4E and FIG. 11E). These Apex epitope-specific BCRs with long HCDR3s primarily contained IGHD3-15 (96%), and more than 88% were Apex bnAb-like according to our definition (FIG. 4F and Table 3). As for the protein group, none of the Apex epitope-specific BCRs met the criteria for the DH3-41 precursor. In total, the inventors identified 218 paired Apex bnAb-like precursor memory BCRs from Apex epitope-specific Bmem cells clustered into 80 unique lineages across all six animals (FIG. 4G). These lineages exhibited high polyclonal diversity. A substantial number of clones65DM2\301045646.1PATENT Docket No. Y7969-99073 possessed very long HCDR3 (length > 26 aa). Among all six animals, the median frequency of Apex bnAb-like precursors among Bmem cells at week 10 was 0.0308% (approximately 1 in 3252), similar to that of the protein immunization group (FIG. 4H).

[0324] When comparing the protein and mRNA-LNP groups further, the inventors observed a higher frequency of ApexGT6++Bmem cells among the total Bmem cells in the protein group (FIG.41). However, among ApexGT6++memory BCRs, the mRNA-LNP group showed a higher percentage of Apex bnAb-like precursors compared to the protein group (FIG. 4J). Hence, under the dosing conditions of our experiments, adjuvanted-protein immunization induced a stronger ApexGT6++Bmem response, whereas the mRNA-encoded membrane-bound platform induced an ApexGT6++Bmem response with superior focusing on the Apex epitope (which was consistent with the serum results). The balance of these factors resulted in protein and mRNA-LNP inducing similar overall frequencies of Apex bnAb-like precursors among Bmem cells.

[0325] The mRNA-LNP immunized animals produced a higher frequency of Apex bnAb-like precursors with very long HCDR3s (length > 26 aa) compared to RMs immunized with protein (FIG. 4K and FIG. 11F). This suggested that mRNA-encoded membrane-bound ApexGT6 might have greater potential to recruit B cells with very long HCDR3s. This feature might be important because, among the human Apex bnAbs, those with longer HCDR3s exhibit greater breadth and potency (FIGs. 12G-H). In summary, two bolus immunizations of mRNA-LNP encoding membrane-bound ApexGT6 induced strong Apex epitope-specific Bmem cell responses in all six animals, with a higher proportion of Apex bnAb-like precursors compared to adjuvanted-protein immunization.Example 7: Functional and Structural Characterization of Elicited Antibodies

[0326] Analysis of somatic hypermutation (SHM) revealed that Apex bnAb-like precursor GC BCRs from the protein immunization group accumulated mutations over time, reaching median values of 6.5% amino acid mutation after the second immunization (FIG. 5 A). The pattern of mutation acquisition differed between heavy and light chains, with VH genes acquiring more SHM after the first immunization, while VK / Z genes showed greater mutation accumulation after the second immunization (FIG. 12A). In the mRNA-LNP group, Apex bnAb-like precursor memory BCRs showed similar median SHM values to the protein group (FIG. 5B), providing evidence for robust germinal center responses despite limited accessibility for direct GC sampling.66DM2\301045646.1PATENT Docket No. Y7969-99073

[0327] To evaluate the functional consequences of these mutations, monoclonal antibodies (mAbs) were generated from selected representative Apex bn Ab-like precursors isolated at various timepoints from different animals. Inferred germline versions (iGL) of each antibody were also produced by reverting all recognizable templated SHMs to germline sequences. Surface plasmon resonance (SPR) analysis demonstrated that Apex bnAb-like precursor mAbs derived from GC BCRs showed progressive improvement in affinity, reaching a median KD of approximately 0.24 nM by week 12 (FIG. 5C). This represented a more than 258-fold increase in affinity compared to their corresponding inferred germline versions (FIG. 12B).

[0328] All tested ApexGT6 binders showed no detectable affinity for ApexGT6. KO, confirming their Apex epitope specificity (FIG. 5C). Those that did not bind to ApexGT6 did not bind to ApexGT6. KO either. Hence, in those cases, although ApexGT6 binding was detected by the original B cell sorting using substantial avidity, the binding was weak enough that it could not be detected by SPR at the highest trimer concentration tested (approximately 10 pM). It remains possible that at least a fraction of these cells were not true ApexGT6 binders but were sorted due to a wide gating strategy. The complete results along with the sequences of the elicited antibodies are shown in Table 5.

[0329] Although strict minimum length criteria were used for defining an Apex bnAb-like HCDR3, based on the known Apex bnAbs, it remains uncertain whether those with HCDR3s longer than 20 aa but shorter than 24 aa have the potential to become Apex bnAbs. A significant percentage of Apex epitope-specific binders after ApexGT6 immunizations had HCDR3 lengths of 22 to 23 aa. This suggested that a 22- or 23-aa HCDR3 was at least long enough to penetrate the N156 / N160 glycan shield and bind to the Apex region of ApexGT6. Furthermore, there were no statistically significant differences in affinity toward the immunogen among these mAbs (FIG.5D). Follow-up studies will be required to understand the potential of Abs with length 22 - 23 aa HCDR3s for bnAb development.

[0330] For memory B cell-derived antibodies, all 60 mAbs identified as Apex bnAb-like precursors by sequence analysis demonstrated specific binding to the Apex epitope (FIG. 5E). The affinity for ApexGT6 at week 10 was similarly strong for mAbs from both immunization groups, with median KD values of 1 nM and 0.2 nM for mRNA and protein groups respectively.

[0331] To assess whether affinity maturation against the ApexGT6 immunogen had resulted in on-track maturation toward bnAb development, binding to more native-like Apex epitopes was67DM2\301045646.1PATENT Docket No. Y7969-99073 evaluated. The apex epitope on the trimer is composed of two main motifs, strand C (48, 49) and V2b loop (18, 50), both of which play a crucial role in antigen-antibody recognition. To improve the affinity to the Apex bnAb-like precursors, these motifs were modified with multiple germlinetargeting mutations in ApexGT6. However, an Apex bnAb should be able to recognize an epitope that has these germline-targeting mutations reverted to the ones most commonly found in HIV strains in global circulation. For the V2b loop (179-191, based on HXB2 sequence numbering), analysis of Env sequences from the Los Alamos database (57) indicated that the most common features include a hydrophilic loop of length 13 to 16 aa with one or two glycosylation sites (FIG.5F and FIG. 12C). The V2b loop of ApexGT6 fulfills the length requirement (13 aa), but includes multiple hydrophobic GT mutations, and lacks glycosylation sites. For strand C, two germlinetargeting mutations, D167N and K169R, were found to have favorable interactions with the germline-reverted variant of PCT64 (18). Sequence analysis indicated that 167D and 169K are the most frequent aa at these two positions among HIV isolates (FIG. 5G and FIG. 12D).

[0332] Accordingly, the affinities of post-primed Apex bnAb-like mAbs and their iGLs wee assessed for variants of the ApexGT6 trimer with either a wild type V2b loop (derived from isolate 191084, referred to as ApexGT6-V2b), or with 167D and 169K (referred to as ApexGT6-DK) on the strand C.

[0333] When testing the post-immunized Apex bnAb-like mAbs against ApexGT6-V2b, the affinity significantly decreased, with only 47% of the iGLs having detectable binding and affinities weaker than 1 pM (FIG. 5H). This decrease in affinity appeared to be caused by reverting the hydrophobic mutations to hydrophilic residues (L187N and W188S), and by adding a glycosylation site atN187 (FIG. 12E). After the second immunization, all tested Apex bnAb-like mAbs derived from GC BCRs were able to bind to ApexGT6-V2b, with a median KD value of approximately 4.9 pM. More than half of the Apex bnAb-like mAbs, derived from memory BCRs, were capable of binding to ApexGT6-V2b for both the mRNA and protein groups at week 10. The median KD values among binders were 1.4 pM and 5.3 pM, respectively.

[0334] Compared to ApexGT6, ApexGT6-DK abolished binding of nearly half of the clones that bound iGL, suggesting that these GT mutations are required for priming these precursors in RMs. The affinity of Apex bnAb-like GC-derived mAbs for the DK trimer increased over time (FIG. 12F). After the second immunization, more than 85% of the tested Apex bnAb-like mAbs derived from GC BCRs showed an affinity stronger than 100 nM for the ApexGT6-DK trimer68DM2\301045646.1PATENT Docket No. Y7969-99073 (FIG. 51). For post-immunized Apex bnAb-like memory-derived mAbs, the affinity to the DK version of ApexGT6 was similarly strong for both the mRNA and protein groups, with median KD values of 164 nM and 558 nM, respectively.

[0335] Cell surface antigenic profiling was also performed to assess the affinity of Apex bnAb-like binders against the membrane-bound ApexGT6 trimer and its DK and WT V2b loop variants. All three Apex trimers exhibited an overall antigenic profile similar to that of MD39, except for increased binding to the non-neutralizing V3-directed mAb 4025 (FIG. 5J). The three ApexGT6-induced Apex bnAb-like mAbs demonstrated strong binding to ApexGT6, but not to membranebound BG505 SOSIP (referred as gp151) or BG505 MD39 (referred as MD39) trimers. REtl8_wkl7_023 and RJzl8_wkl2_041 showed good binding to ApexGT6-V2b, while RIol8_wk8_018 and RJzl8_wkl2_041 showed good binding to ApexGT6-DK. Taken together, these findings suggested that two doses of ApexGT6 immunization induced Apex bnAb-like precursor B cells with SHMs that conferred the ability to recognize apex epitopes closer to native on soluble protein and membrane-bound trimers, providing evidence for vaccine-induced maturation toward bnAb development.Example 8: Structural Analysis of Elicited Antibodies

[0336] To investigate how the Apex bnAb-like BCRs induced by ApexGT6 recognize the apex, the inventors determined cryoEM structures of two high affinity binders, RJzl8_wkl2_038 (referred to as RM038) and RIol8_wk8_018 (referred to as RM018), in complex with ApexGT6.

[0337] The RM038 liganded structure showed the expected 1:1 stoichiometry and a tilted angle of approach relative to the 3-fold axis with extensive interactions to N 160 andN156 glycans, similar to other Apex bnAbs and their inferred germlines (18, 52) (FIG. 6A). From a top-down view, RM038 used its extended long HCDR3 to bind to the center of the apex of ApexGT6, similar to the of PCT64 LMCA to ApexGT2 (18), but with heavy and light chains flipped (FIGs. 6, B-C). The aspartic acids within the DDY motif interacted with the residues around the top surface of the 3-fold axis (R166, N167, andR169, FIG. 13A), similar to the counterparts on PCT64 LMCA (FIG.13B). However, the HCDR3 of RM038 was less extended towards the 3-fold axis than PCT64 LMCA. Notably, the sulfated tyrosine within DDY motif of RM038 interacted around the top surface of the 3-fold axis, including V127, G128, and the base of the N160 glycan on the same protomer (FIG. 13C), instead of being buried inside like PCT64 LMCA, which interacted with T123 and P124 (FIG. 13D). Rotation of the structure by 90° revealed that the HCDR3 of RM03869DM2\301045646.1PATENT Docket No. Y7969-99073 formed a second lobe that interacted to strand C of one of the protomers (FIGs. 6D-E). Thus, the HCDR3 of RM038 had similarities to the hammer-head motif on the HCDR3 of the Apex bnAb PG9 (FIG. 13E), but with the heavy and light chains flipped when RM038 is aligned with PG9 liganded structures (75) (FIG. 13F). Overall, the HCDR3 of RM038 exhibited hybrid features of PCT64 and PG9 when interacting with the apex epitope of ApexGT6 (FIG. 6, C-E). In addition to the protruding HCDR3 interacting at the center of the apex, RM038 also had favorable interactions with the highly engineered V2b loop of ApexGT6. The HCDR2, the upper stem of the HCDR3, and the LCDR3 formed a hydrophobic pocket (FIG. 6F and FIG. 13G), facilitating numerous favorable interactions with the engineered hydrophobic V2b loop on the trimer immunogen. The PCT64 LMCA interaction with ApexGT2 exhibited similar counterparts in its HCDR3 and HCDR1 (18). Several polar and acidic SHMs on HCDR1 and HCDR2 might enhance binding to the N160 glycans (FIG. 6G), which commonly occurred during Apex bnAb maturation (49, 52-55).

[0338] The structure of the second mAb (RM018) in complex with ApexGT6 also showed the expected 1: 1 stoichiometry and a tilted angle of approach relative to the 3-fold axis with extensive interactions to N160 and N156 glycans (FIG. 7A), with a similar heavy and light chain binding mode as RM038 (FIG. 14A). The heavy and light chains also formed a hydrophobic pocket around V2b mutations as observed for RM038 (FIG. 7B). The HCDR3 of RM018 formed an extended loop that interacted with strand C on one protomer of the trimer and with the DDY motif at the tip of the long HCDR3 (FIG. 7C). Unlike PCT64 LMCA or RM038, the DDY tip on the HCDR3 of RM018 interacted around the Apex 3-fold axis in a more parallel manner, with the tyrosine within the DDY motif being unsulfated (FIGs. 14B-C). This binding mode is similar to the Apex bnAb CH01-04 (54) (FIGs. 7D-E). Beyond the HCDR3s, RM018 also exhibited extensive glycan interactions with the heavy chain framework regions (FR) 2 and 3, as well as CDR2s, similar to CH04 but with the heavy chain and light chain positions reversed (FIGs. 7F-G).

[0339] In a previous study (18), the CHOI -04 class of bnAbs was not prioritized for Apex vaccine design due to its low precursor frequency arising from its use of a rare Jn gene (JH2). TO revisit this question, the inventors calculated that CH01-04-class BCRs utilizing a more common JH gene (JH4) would have a substantially higher precursor frequency (median frequency of 0.30 per million naive B cells in 13 out of 14 donors, FIG. 7H) compared to using JH2 (median frequency of 0.09 per million naive B cells in 2 out of 14 donors). This frequency surpasses that70DM2\301045646.1PATENT Docket No. Y7969-99073 of PG9 / PG16, which were previously considered to have the second-highest precursor frequency in the human immunoglobulin repertoire among long HCDR3 -dependent V2 apex bnAbs (FIG.7H). The inventors then produced variant CH01-04-like antibodies using the more common JH4 gene and evaluated their neutralization capacity. The inventors found that the Jn4-variant of CH04 had slightly improved neutralization breadth and potency (74% breadth with a geometric mean IC50 of detected 0.39 pg / mL) compared to WT CH04 (70% breadth with a geometric mean IC50 of 0.53 pg / mL) (FIG. 71). This was tested over a panel of 23 viruses, including strains sensitive to CH01-04 (FIG. 14D). Therefore, the inventors propose that the Ind-variant CH01-04-class represents an attractive target for vaccine induction. Eliciting a CHOI -04 response in addition to a PCT64-class response would potentially expand the breadth of neutralization induced, because CH01-04 can neutralize some HIV strains that PCT64 cannot. It is not entirely surprising that ApexGT6 can elicit a CH01-04-like response in NHPs, given that it can bind to the iGLs and a human NGS precursor of CH04 with appreciable affinty (FIG. 7J and FIG. 14E). The inventors concluded that Jn4-variant CHOI -04 bnAbs are an important vaccine target and ApexGT6 has the potential to prime precursors for this class of bnAb.

[0340] The structural analyses revealed that Apex bnAb-like BCRs induced by ApexGT6 exhibited structural features similar to three human Apex bnAbs. This suggested that ApexGT6 might be capable of eliciting multiple Apex bnAb-related responses simultaneously, potentially leading to broader neutralization coverage through complementary recognition modes.Example 9: Detailed Characterization of ApexGT Immunogen Design and Properties

[0341] Expanding on the immunogen development process, extensive characterization was performed to validate the design and properties of ApexGT6 and its variants. SPR analysis demonstrated that ApexGT6 showed enhanced affinity for multiple PCT64 and PG9 precursors identified from next-generation sequencing of HIV seronegative individuals (FIG. 8A). The binding data for these precursors, including Pre.15 (PG9-like NGS precursor) and Pre.7 (PCT64-like NGS precursor), revealed significant improvements compared to previous designs.

[0342] Antigenic profiling by ELISA demonstrated that ApexGT6 maintained proper trimer conformation while incorporating the desired germline-targeting features (FIG. 8B). The profiles of MD39, ApexGT5, and ApexGT6 trimers were assessed using a panel of antibodies including:• Closed-trimer binding bnAbs (PGT151, PGT145, PG9, PCT64.35S)• Protomer-binding bnAbs (PGT121, 12A12)71DM2\301045646.1PATENT Docket No. Y7969-99073 • Open-trimer binding non-nAbs (4025, B6, Fl 05)

[0343] Thermal stability analysis by differential scanning calorimetry revealed excellent stability for all ApexGT variants (FIG. 8C), with melting temperatures of:• ApexGT5: 72.36°C• ApexGT5.congly: 72.58°C• ApexGT6: 71.48°C• ApexGT6.congly: 70.91°C

[0344] Comprehensive glycosylation profiling using DeGlyPHER provided detailed characterization of site occupancy throughout the trimer (FIG. 8D). This analysis demonstrated proper glycosylation at key sites, including the conserved N-linked glycans critical for apex epitope formation. The glycosylation profile comparison between BG505 MD39, ApexGT6 congly, and ApexGT6 L14 revealed consistent patterns at important sites while confirming the intended modifications at positions N241 and N289.

[0345] The development of precise criteria for identifying Apex bnAb-related precursors was critical for subsequent analyses (FIG. 8E). These criteria were established based on detailed examination of known Apex bnAbs and included specific requirements for:• HCDR3 length• D gene usage and positioning• Presence of key motifs (e.g., DDY)• Positioning of critical residues within the HCDR3

[0346] Analysis of D gene frequencies among human DDY-containing sequences revealed a distinct pattern of usage (FIG. 8F), while examination of the RM repertoire showed enrichment of IGHD3-15 among DDY-containing sequences, supporting the relevance of the animal model for studying these responses.Example 10: Detailed Analysis ofB Cell Responses and Flow Cytometry Characterization

[0347] The analysis of serum responses was extended beyond basic binding studies to provide detailed characterization of epitope specificity and comparative responses between immunization groups. ELISA quantification of serum IgG titers revealed distinct patterns of antigen recognition between ApexGT6 and control immunogens (FIG. 9A). When comparing binding to the intact immunogen (labeled as “live”) versus the Apex-binding knockout variant (labeled as “KO”), clear72DM2\301045646.1PATENT Docket No. Y7969-99073 epitope-specific responses were observed following ApexGT6 immunization but not with the control MD39 immunogen.

[0348] The importance of germline-targeting modifications was further demonstrated through composite analysis of polyclonal responses using negative- stain EM (FIG. 9B). Animals immunized with MD39 showed markedly different antibody targeting patterns compared to ApexGT6-immunized animals, with minimal recognition of the apex epitope region.

[0349] Flow cytometry analysis and cell sorting employed a sophisticated multi-parameter strategy to identify and isolate Apex-specific B cells. For germinal center B cells, the gating strategy (FIG. 9C) included:1. Initial identification of CD20+CD38’ cells2. Dual-color ApexGT6 probe staining (BV650 and BV421)3. Negative selection using ApexGT6. KO PE probe4. Sorting of CD20+CD38 ApexGT6++cells for BCR sequencing

[0350] A parallel but distinct strategy was employed for memory B cells (FIG. 9D):1. Identification of CD20+IgD‘ cells2. ApexGT6 probe staining using AF647 and BV4213. Counter-selection with ApexGT6. KO PE4. Isolation of CD20+IgD' ApexGT6++cells

[0351] The sorting strategy was validated through post-sort analysis and demonstrated consistent identification of apex-specific cells across multiple animals and timepoints. Quality control measures included:• Assessment of probe specificity using control antibodies• Validation of binding patterns using multiple fluorophore combinations• Consistency checks across technical replicates• Verification of sorted population purity

[0352] This rigorous flow cytometry approach enabled reliable identification and isolation of apex-specific B cells for subsequent molecular and functional analyses, while minimizing potential contamination with non-specific or cross-reactive cells. The consistency of the sorting strategy across animals and timepoints facilitated reliable comparative analyses between different immunization groups and temporal tracking of response development.73DM2\301045646.1PATENT Docket No. Y7969-99073 Example 11: Comprehensive BCR Sequence Analysis and Clonal Lineage Characterization

[0353] Single B cell receptor sequencing was performed using a systematic approach outlined in FIG. 10A. Following isolation of ApexGT6++B cells, comprehensive sequencing analysis yielded paired heavy and light chain sequences that were subjected to detailed bioinformatic analysis. The sequencing depth and recovery varied by compartment and timepoint, with a total of 8,168 ApexGT6-negative and 2,376 ApexGT6++paired BCR sequences obtained from FNA samples, and 678 ApexGT6++sequences from PBMC samples in the protein immunization group.

[0354] HCDR3 length distribution analysis revealed distinct patterns between responding and non-responding populations (FIG. 10B). Each animal's repertoire was analyzed individually to account for potential animal-to-animal variation. For example, animal REtl8 showed a clear enrichment of long HCDR3s among ApexGT6++cells compared to ApexGT6- cells, with the enrichment being even more pronounced among epitope-specific cells. Similar patterns were observed across multiple animals, though with some variation in the exact distribution of HCDR3 lengths.

[0355] A critical finding emerged from the analysis of IGHD gene usage among long HCDR3-containing sequences (FIG. 10C). Three distinct populations were compared:

[0356] ApexGT6‘ GC BCRs with long HCDR3s (>24 amino acids)

[0357] ApexGT6++GC BCRs with long HCDR3 s

[0358] Epitope-specific GC BCRs with long HCDR3s

[0359] The analysis revealed progressive enrichment of IGHD3-15 usage, with the highest frequency observed among epitope-specific sequences. This enrichment was particularly striking when compared to the baseline distribution of IGHD genes in naive rhesus macaque B cells.

[0360] Clonal lineage analysis was performed using stringent criteria to identify related sequences while avoiding artificial clustering. Key parameters included:• Identical V and J gene usage• Highly similar HCDR3 lengths (±1 amino acid)• HCDR3 sequence similarity above a defined threshold• Shared somatic mutations indicating common ancestry

[0361] This analysis revealed multiple distinct lineages across animals, with varying degrees of expansion and somatic hypermutation. Several notable features emerged from the lineage analysis:74DM2\301045646.1PATENT Docket No. Y7969-99073 1. Convergent features across independent lineages:• Common motifs in HCDR3 sequences• Similar patterns of somatic hypermutation• Shared structural solutions for apex recognition2. Temporal development patterns:• Progressive accumulation of mutations• Maintenance of key binding motifs• Evolution of affinity-enhancing changes3. Individual lineage characteristics: Each lineage was analyzed for specific features that contributed to apex recognition. For example, lineages from animal RJzl8 showed distinct patterns of HCDR3 evolution while maintaining the core DDY motif. The diversity of lineages demonstrated the ability of ApexGT6 to engage multiple precursor B cells with different starting sequences but similar structural solutions for epitope recognition.

[0362] Further analysis of lineage development revealed complex patterns of evolution across the immunization period. Sequences within each lineage showed evidence of ongoing selection, with maintenance of key structural elements while accumulating affinity-enhancing mutations. Many lineages demonstrated convergent evolution despite emerging from different precursors, suggesting that the immunogen successfully guided multiple B cell populations toward similar structural solutions for apex recognition.Example 12: Comparative Analysis ofmRNA and Protein Immunization Platforms

[0363] The mRNA-LNP immunization platform demonstrated distinct advantages in focusing the immune response toward the apex epitope. ELISA analysis revealed that while protein immunization generated higher overall binding titers, the mRNA platform produced responses with superior epitope specificity (FIG. 11 A). This was particularly evident when comparing the ratio of apex-specific to total ApexGT6 binding, where mRNA immunization consistently showed higher specificity despite lower absolute titers (FIG. 1 IB).

[0364] A detailed comparison of serum responses between MD39 and ApexGT6 immunization groups revealed clear differences in epitope targeting (FIG. 11C). The ApexGT6 mRNA group showed significantly higher apex-specific responses compared to both the MD39 mRNA group and the protein immunization groups. This enhanced focusing effect was maintained even when analyzing responses to more native-like forms of the immunogen.75DM2\301045646.1PATENT Docket No. Y7969-99073

[0365] Memory B cell analysis in the mRNA immunization group revealed distinct population dynamics compared to protein immunization. The percentage of ApexGT6++Bmem cells showed consistent increases following each immunization, with maintenance of high epitope specificity throughout the response (FIG. 11D). HCDR3 length distribution analysis of memory BCRs showed a striking enrichment of longer sequences, particularly among apex-specific cells (FIG. 11E).

[0366] One of the most notable features of the mRNA platform was its ability to induce a higher proportion of very long HCDR3s (>26 amino acids) compared to protein immunization (FIG. 11F). This characteristic proved particularly relevant given the correlation between HCDR3 length and neutralization breadth observed among natural apex bnAbs (FIGs. 11G-H). The ability to preferentially expand B cells with longer HCDR3s suggests that the mRNA platform may offer advantages for targeting difficult-to-access epitopes requiring extended antibody structures. Example 13: Detailed Characterization of Antibody Maturation and Affinity Development

[0367] The maturation of vaccine-induced responses was characterized through detailed analysis of somatic hypermutation patterns and evolution of binding properties. Sequential analysis of mutations in heavy and light chain genes revealed distinct temporal patterns of affinity maturation. The heavy chain variable regions showed rapid accumulation of mutations following the primary immunization, with a median of 6.5% amino acid changes by week 12 (FIG. 12A). In contrast, light chain mutation accumulation showed a more gradual pattern, with significant increases observed primarily after the second immunization.

[0368] Affinity measurements of monoclonal antibodies isolated at different timepoints provided crucial insights into the functional consequences of somatic hy permutation. When compared to their inferred germline counterparts, mature antibodies showed dramatic improvements in binding strength, with some clones demonstrating more than 258-fold increases in affinity (FIG. 12B). This enhancement in binding properties correlated with the accumulation of specific mutations in both CDR and framework regions.

[0369] Analysis of HIV Env sequences from the Los Alamos database provided critical context for evaluating the maturation pathway toward recognition of native viral epitopes. The V2b loop region showed considerable natural variation in both length and glycosylation patterns (FIG. 12C). The most common features included hydrophilic loops of 13-16 amino acids76DM2\301045646.1PATENT Docket No. Y7969-99073 containing one or two N-linked glycosylation sites. Similarly, analysis of strand C revealed strong conservation of specific residues, particularly at positions 167 and 169 (FIG. 12D).

[0370] The impact of specific mutations on antibody recognition was systematically evaluated using a panel of ApexGT6 variants. Reversion of hydrophobic residues in the V2b loop to their hydrophilic counterparts, coupled with introduction of glycosylation sites, revealed the structural basis for decreased affinity to more native-like forms (FIG. 12E). However, antibodies isolated after the second immunization showed improved ability to accommodate these changes, suggesting evolution toward broader recognition properties.

[0371] Temporal analysis of binding to the ApexGT6-DK variant demonstrated progressive improvement in recognition of more native-like features (FIG. 12F). Early antibodies showed strong dependence on germline-targeting mutations, but later variants acquired the ability to recognize more native-like epitope conformations while maintaining high affinity. This evolution pattern suggests that the immunization strategy successfully guided antibody development toward features required for recognition of native viral spikes.Example 14: Structural Analysis ofRM038 and Related Antibodies

[0372] To investigate the structural characteristics of RM038 and its capacity to bind to the HIV apex epitope, structural analyses were conducted, including cryo-electron microscopy (Cryo-EM), to elucidate binding motifs and HCDR3 structural adaptations.

[0373] Cryo-EM analysis was performed on RM038 in complex with the HIV Env trimer ApexGT6, utilizing a data collection strategy and processing pipeline similar to previously described protocols for apex-targeting bnAbs (e.g., PCT64, PG9). The structure revealed that RM038 features an extended HCDR3 loop, measuring 26 amino acids, which penetrates the glycan shield and forms specific contacts with conserved residues in the strand C of the apex epitope (FIG.13 A).

[0374] Further comparisons were conducted between RM038 and previously studied apextargeting antibodies, including PG9 and PCT64, highlighting RM038’s unique structural adaptations that enhance its breadth and potency.Table 4. Neutralization Analysis of Antibody VariantsVirus Strain IC50 (ug / niL)CH04 CH04. JH4 PCT64.35S PG9 PGDM140077DM2\301045646.1PATENT Docket No. Y7969-99073 TRO 11 0.36 0.040 0.082 0.11 0.07725710 0.85 0.24 0.025 0.018 0.0024398F1 0.17 0.25 NN 26 NNCNE8 NN NN 1.0 0.091 0.0023X2278 0.055 0.038 2.3 0.0056 0.0080BJQX002000 5.6 1.8 0.15 0.042 0.0067X1632 44 20 0.50 0.11 0.0072CE1176 0.053 0.034 NN 0.0070 0.0074246F3 0.33 1.08 0.022 0.011 0.0017CH119 0.87 0.74 0.4 0.4 0.064CE0217 0.12 0.075 0.46 0.003 0.0015CNE55 NN 16.8 0.4 0.35 0.005092RW020 NN NN NN 0.40 0.01094UG103 8.6 7.2 0.76 0.38 0.010BGN 0.18 0.054 0.044 0.018 0.002892BR020 NN NN NN NN NNJRCSF 0.26 0.31 0.022 0.0036 0.0036JR. FL NN NN NN NN NNWITO 0.073 0.023 0.0211 0.012 0.0035IAVIC22 NN NN NN 0.096 NN93IN905 NT NT NT NT NT16055 9.3 4.2 0.17 0.0086 0.0010492TH021 0.11 0.089 0.041 0.13 0.0019MLV NN NN NN NN NNGeomean* 0.53 0.39 0.15 0.06 0.01% detected 70 74 70 87 78NN = No neutralization detected; NT = Not tested; *Geometric mean calculated for detected values only.

[0375] High-Resolution Structural Analysis of RM038 The cryo-EM analysis achieved an overall resolution of 3.2 A, enabling detailed characterization of key binding interactions. Data collection utilized a 200 kV Glacios microscope equipped with a Falcon 4 detector, with approximately 5,177 micrographs collected. The final reconstruction incorporated 131,958 molecular projection images processed using CryoSPARC Live followed by comprehensive refinement in CryoSPARC 3.2 and 4.4.

[0376] The complex structure revealed that RM038 engages the apex epitope through a sophisticated network of interactions. The antibody's HCDR3 adopts a two-lobed structure, with one lobe penetrating toward the 3-fold axis while the second lobe forms extensive contacts with strand C.78DM2\301045646.1PATENT Docket No. Y7969-99073

[0377] The binding mode demonstrates remarkable convergence with known bnAbs while utilizing unique structural features that enhance affinity and specificity.Example 15: Structural and Functional Analysis ofRM018 and CH01-04-related Responses

[0378] To extend the structural and functional insights of RM038, further analysis was conducted on RM018 and CH01-04-related responses to evaluate their binding profiles and neutralization potency across diverse HIV clades. RM018, like RM038, demonstrated a long HCDR3 loop (>24 amino acids) optimized for apex epitope engagement, while CH01-04-related antibodies, with shorter HCDR3s, required additional somatic hypermutations to achieve comparable neutralization potency.

[0379] Structural assessment of RM018 in complex with the ApexGT6 trimer was performed using Cryo-EM, which provided a resolution of 3.2 A (FIG. 14A). RM018’sHCDR3 loop interacts with the apex through a distinct DDY motif positioned centrally within the loop, allowing targeted binding to glycan N156.

[0380] Comparative Structural Analysis of Apex Recognition Modes The structural analysis of RM018 and CH01-04-related antibodies revealed three distinct recognition modes for targeting the apex epitope:

[0381] The co-existence of these recognition modes suggests multiple viable paths to achieving broad neutralization, with implications for vaccine design strategies.Example 16: Advanced Characterization of Antibody Maturation Pathways

[0382] Comprehensive analysis of antibody maturation pathways was performed by integrating sequence, structural, and functional data from immunized animals. The evolution of apex-targeting responses was tracked through detailed examination of somatic hypermutation patterns, structural adaptations, and affinity measurements at multiple timepoints.

[0383] Sequential sampling revealed distinct phases of antibody evolution:

[0384] Phase 1 (Weeks 0-4): Initial responses showed preferential expansion of B cells utilizing IGHD3-15, with minimal somatic hypermutation (median 2.1%). These early antibodies demonstrated strong dependence on germline-targeting mutations in ApexGT6, with KD values typically above 100 nM.79DM2\301045646.1PATENT Docket No. Y7969-99073

[0385] Phase 2 (Weeks 4-8): Heavy chain maturation dominated this period, with focused mutations in HCDR1 and HCDR2 regions improving glycan accommodation. Affinity enhancement was modest (median KD ~50 nM), but recognition of more native-like features began to emerge.

[0386] Phase 3 (Weeks 8-12): Light chain maturation accelerated following the boost immunization, with particular focus on LCDR3 modifications that enhanced binding stability. The most evolved antibodies achieved KD values below 1 nM for ApexGT6 while maintaining recognition of more native-like epitope variants.Example 17: Materials and Methods

[0387] Cell Lines and Culture Conditions

[0388] Study design

[0389] This study primarily aimed to assess whether germline-targeting vaccination could consistently elicit bnAb-like precursor responses targeting the HIV Env Apex region in outbred primates. To achieve this goal, we immunized rhesus macaques with ApexGT6, a germlinetargeting trimer with affinity for additional PCT64- and PG9-class precursors. We administered it in two forms: as an adjuvanted soluble protein or as an mRNA-encoded membrane-anchored protein. Throughout the study, we assessed epitope-specific responses in serum, germinal centers, and memory cells. Binding assays were conducted to examine epitope specificity and acquired affinity maturation through immunization. Structural analyses were performed to investigate how the macaque Apex bnAb-like BCRs induced by ApexGT6 recognize the Apex epitope, and to validate whether they exhibited HCDR3 features similar to human Apex bnAbs. For animal experiments, the investigators were not blinded to the group identities.

[0390] Cell lines

[0391] This study used HEK 293F cells, for production of soluble proteins in suspension and for cell-surface expression and antigenicity testing; HEK 293T cells (ATCC) were used to produce viruses; rtTA3G-expressing HEK 293T cells were used for mammalian display directed evolution.

[0392] For virus production, HEK 293T cells (ATCC) were maintained in complete DMEM (Gibco), supplemented with 10% FBS, 2 mM L-glutamine (Gibco), and 1% Pen-Strep. TZM-bl cells (NIH AIDS Reagents Program) were maintained in complete DMEM and used as target cells in pseudovirus neutralization assays.80DM2\301045646.1PATENT Docket No. Y7969-99073

[0393] For mammalian display, HEK 293T cells were cultured at 37°C in Advanced DMEM (Gibco) supplemented with 5% FBS (Gibco), GlutaMAX (Gibco), 2-mercaptoethanol (Gibco) and Antibiotic-Antimycotic (Gibco); rtTA3G-expressing HEK 293T cells were cultured under the same conditions but with the addition of 10 pg / mL blasticidin (Gibco).

[0394] All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2. Sex of both cell lines was female. Cell lines have not been authenticated.

[0395] DNA gene synthesis and cloning

[0396] Five types of gene constructs were used in this study. Recombinant trimers and antibodies were synthesized at GenScript, Inc. Trimers were cloned into pCW between the signal sequence and a C-terminal GTKHHHHHH (SEQ ID NO: 14) tag using the Agel and Kpnl cloning sites. Trimer probes were cloned into pHLsecAvi2 between the signal sequence and a C-terminal flexible linker (GTGGSGGSG) (SEQ ID NO: 15), followed by an Avi tag (LNDIFEAQKIEWHE) (SEQ ID NO: 16) connected through a flexible linker with a his tag (GGSGGSFOTHHHH) (SEQ ID NO: 17) using the Agel and Kpnl cloning sites. Antibody heavy chains were cloned into pCW-CHIg-hGl between the leader and the IgGl human constant domain using the EcoRI and Ndel cloning sites. Kappa chains were cloned into pCW-CLIg-hk between the leader and the human kappa constant region using the EcoRI and BsiWI cloning sites. Lambda chains were cloned into pCW-CLIg-hL2 between the leader sequence and the last aa in lambda FW4 using the EcoRI and AvrII cloning sites.

[0397] Genes used for library design were cloned into pENTR via Gibson assembly. This included a C-terminal cMyc epitope followed by a platelet-derived growth factor receptor (PDGFR) transmembrane domain. Error-prone PCR libraries were constructed using the GeneMorph II Random Mutagenesis Kit (Agilent). Combinatorial NNK libraries were ordered as ultramers from IDT, using ambiguous nucleotides at the combinatorial positions. Alternating complement and reverse complement ultramers were assembled with outer primers, overlapping an insertion site by 30 bp.

[0398] ApexGT immunogen design

[0399] Lentiviral mammalian display and directed evolution were performed as described previously (Steichen et al 2016), with modifications to sorting probes and libraries. The epPCR library, starting with the BG505. ApexGT2 based construct, introduced random mutations at positions 86-229 (HXBC2 numbering). The combinatorial NNK library, starting with the81DM2\301045646.1PATENT Docket No. Y7969-99073 BG505. ApexGT5 based construct, had three combinatorial NNK positions K121, T128, and K168 using IDT.

[0400] Cells were incubated with PCT64 LMCA IgG and B6 Fab with a V5 tag, washed with FACS buffer, and then stained with fluorescein isothiocyanate (FITC)-labeled a-cMyc (Immunology Consultants Laboratory), R-Phycoerythrin AffiniPure F(ab')2 Fragment Goat AntiMouse IgG, Fey fragment specific (Jackson), and V5-Tag Antibody | SV5-Pk1 (Bio-Rad). Cells were sorted on a BD Influx (BD Biosciences) FACS sorter. Approximately 10K of the desired gated cells were collected and expanded for ~ one week in the presence of puromycin and blasticidin before the next round of enrichment was carried out. The genomic DNA for the final enriched libraries was extracted and PCR amplified using partial adapters recommended by GeneWiz “EZ amplicon”. We determined enriched mutations as described previously (Steichen et al 2016).

[0401] ApexGT protein expression

[0402] BG505-based ApexGT variants were cloned into pCW as described above and transformed into DH5a cells following the manufacturer's protocol. Plasmids were maxi-prepped using a BenchPro 2100 (Thermo Fisher Scientific). Trimer plasmids were co-transfected with furin in a 2:1 ratio into 293F cells cultured in FreeStyle media (Life Technologies) using polyethylenimine (PEI, Polysciences, Inc.; 1:3 DNA: PEI ratio) as a transfection reagent in Opti-MEM™ reduced serum medium (Thermo Fisher Scientific, Cat# 31985070). Proteins were harvested from the supernatant after 7 days of incubation at 37°C. For non-tagged trimers, proteins were purified through lectin affinity chromatography using 7.5 mL of lectin beads from Vector Laboratories (#AL-1243) per IL of protein supernatant. This was done using a disposable plastic column from Bio-Rad (#732-1010). Trimer fractions were further isolated using size exclusion chromatography (SEC) on a Superdex 200 PG SEC column (Cytiva, Cat 28-9893-35) on an AKTA Pure 25L HPLC. For his-tagged trimers, proteins were purified by Ni++ affinity chromatography followed by SEC. For biotinylated probes, proteins were expressed with a his-tag and avi-tag, purified by Ni++ affinity chromatography followed by SEC, and biotinylated using a BirA biotinprotein ligase reaction kit (Avidity, Cat# BirA500) according to the manufacturer's instructions. The oligomeric state of the trimers was then confirmed by size exclusion chromatography-multi-angle light scattering using the DAWN HELEOS II multi-angle light scattering system with82DM2\301045646.1PATENT Docket No. Y7969-99073 Optilab T-rEX refractometer (Wyatt Labs). The final proteins were diluted in lx TBS and stored at -80°C.

[0403] Antibody expression and purification

[0404] Paired HC and LC variable region sequences from selected RM BCRs were gene synthesized and inserted into human IgGl constant region expressing vector pCW-CHIg-hGl and human kappa or lambda expressing vectors pCW-CLig-hk or pCW-CLig-hL2, respectively. Heavy and light chains along with TPST1 were transfected with PEI at a 3: 1: 1: 1 PEI:heavy:light: TPSTl ratio into Freestyle 293F cells. Supernatants were harvested on day 7, and batch binding occurred overnight at 4°C with Protein A resin (Thermo Fisher Scientific, Cat# 20334), on a rocker. After elution with IgG elution buffer (0.1 M Glycine pH 2.7, Thermo Fisher Scientific), antibodies were buffer-exchanged into 1 x PBS and then concentrated using a 50k MWCO concentrator (Millipore).

[0405] Surface plasmon resonance (SPR)

[0406] We measured kinetics and affinity of antibody-antigen interactions on Carterra LSA using CMDP Sensor Chip (Carterra) and lx HBS-EP+ pH 7.4 running buffer (20x stock from Teknova, Cat. No H8022) supplemented with BSA at Img / ml. We followed Carterra software instructions to prepare chip surface for ligands capture. In a typical experiment about 500-700 RU of capture antibody (SouthernBiotech Cat no 2047-01) in 10 mM Sodium Acetate pH 4.5 was amine coupled. The critical detail here was the concentration range of the amine coupling reagents and capture antibody. We used N-Hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) from Amine Coupling Kit (GE order code BR-1000-50). As per kit instruction 22-0510-62 AG the NHS and EDC should be reconstituted in 10 ml of water each to give 11.5 mg / ml and 75 mg / ml respectively. However, the highest coupling levels of capture antibody were achieved by using 10 times diluted NHS and EDC during surface preparation runs. Thus, in our runs, the concentrations of NHS and EDC were 1.15 mg / ml and 7.5 mg / ml. The activation time was reduced to 1min. The concentrated stocks of NHS and EDC could be stored frozen in -20°C for up to 2 months without noticeable loss of activity. The SouthernBiotech capture antibody was used at concentration 25ug / ml with 10 minutes contact time. Phosphoric Acid 1.7% was our regeneration solution with 60 seconds contact time and injected three times per each cycle. Solution concentration of ligands was around 1 ug / ml and contact time was 5 min. Raw sensorgrams were analyzed using Kinetics software (Carterra), interspot and blank double referencing, Langmuir model. Analyte concentrations were quantified83DM2\301045646.1PATENT Docket No. Y7969-99073 on NanoDrop 2000c Spectrophotometer using Absorption signal at 280 nm. For best results, analyte samples should be buffer exchanged into the running buffer using dialysis. We typically cover broad range of affinities in our runs and the best referencing practices are different depending on how fast the off-rate is for particular ligand. For fast off-rate (faster than 9xl0-31 / s), we use automated batch referencing that includes overlay y-align and higher analyte concentrations. For slow off-rates (9x10'31 / s or less), we use manual process referencing that includes serial y-align and lower analyte concentrations. After automated data analysis by Kinetics software, we also did additional filtering to remove datasets with highest response signals smaller than signals from negative controls. This additional filtering could be performed automatically using R-script.

[0407] Antigenic profile with enzyme-linked immunosorbent assay (ELISA)

[0408] 96-well plates were coated overnight at 4°C with 6x-His (SEQ ID NO: 18) Epitope Tag Rabbit Antibody (Genscript, Cat. No. A00174) at 2ug / mL in PBS. Plates were washed 3 times with PBS, 0.2% Tween (PBS-T), and blocked with 5% milk and 1%FBS in PBS-T for Ih. Subsequently, 2ug / mL of the purified His-tagged trimers was added for 2h in PBS, after which the plates were washed three times with PBS-T. Serial dilutions of antigenic profiling mAbs in 1% FBS PBS-T were added to the plates for 1 h, after which the plates were washed again three times with PBS-T before the addition of anti-human conjugated peroxidase at 1:5000 for 1 h. After four final washes, binding was detected by the addition of TMB substrate and measured by absorbance at 450 and 570 nm. Background subtraction was performed by subtracting the 570 nm value from the corresponding 450 nm value. Data were subsequently analyzed in Prism (Prism vl0.2; GraphPad Software).

[0409] Differential scanning calorimetry (DSC)

[0410] DSC experiments were performed on a MicroCai VP-Capillary differential scanning calorimeter (Malvern Instruments). The HBS buffer was used for baseline scans and the protein samples were diluted into HBS buffer to adjust to 0.25 mg / mL. The system was allowed to equilibrate at 20°C for 15 min and then heat up till 90°C at a scan rate of 90°C / h. Buffer correction, normalization, and baseline subtraction were applied during data analysis using Origin 7.0 software. The non-two-state model was used for data fitting.

[0411] Glycan analysis

[0412] DeGlyPHER (39) is used to ascertain site-specific glycan occupancy and processivity on the examined glycoproteins.84DM2\301045646.1PATENT Docket No. Y7969-99073

[0413] Proteinase K treatment and deglycosylation: HIV Env glycoprotein was exchanged to water using Microcon Ultracel PL-10 centrifugal filter. Glycoprotein was reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HC1) and alkylated with 10 mM 2-Chloroacetamide in 100 mM ammonium acetate for 20 min at room temperature (RT, 24°C). Initial protein-level deglycosylation was performed using 250 U of Endo H for 5 pg trimer, for 1 h at 37°C. Glycoprotein was digested with 1:25 Proteinase K (PK) for 30 min at 37°C. PK was denatured by incubating at 90°C for 15 min, then cooled to RT. Peptides were deglycosylated again with 250 U Endo H for 1 h at 37°C, then frozen at -80°C and lyophilized. 100 U PNGase F was lyophilized, resuspended in 20 pl 100 mM ammonium bicarbonate prepared in H218O, and added to the lyophilized peptides. Reactions were then incubated for 1 h at 37°C, subsequently analyzed by LC-MS / MS.

[0414] LC-MS / MS: Samples were analyzed on an Q Exactive HF-X mass spectrometer. Samples were injected directly onto a 25 cm, 100 pm ID column packed with BEH 1.7 pm C18 resin. Samples were separated at a flow rate of 300 nL / min on an EASY-nLC 1200 UHPLC. Buffers A and B were 0.1% formic acid in 5% and 80% acetonitrile, respectively. The following gradient was used: 1-25% B over 160 min, an increase to 40% B over 40 min, an increase to 90% B over another 10 min and 30 min at 90% B for a total run time of 240 min. Column was reequilibrated with solution A prior to the injection of sample. Peptides were eluted from the tip of the column and nanosprayed directly into the mass spectrometer by application of 2.8 kV at the back of the column. The mass spectrometer was operated in a data-dependent mode. Full MS1 scans were collected in the Orbitrap at 120,000 resolution. The ten most abundant ions per scan were selected for HCD MS / MS at 25 NCE. Dynamic exclusion was enabled with exclusion duration of 10 s and singly charged ions were excluded.

[0415] Data Processing: Protein and peptide identification were done with Integrated Proteomics Pipeline (IP2). Tandem mass spectra were extracted from raw files using RawConverter (65) and searched with ProLuCID (66) against a database comprising UniProt reviewed (Swiss-Prot) proteome for Homo sapiens (UP000005640), UniProt aa sequences for Endo H (P04067), PNGase F (Q9XBM8), and Proteinase K (P06873), aa sequences for the examined proteins, and a list of general protein contaminants. The search space included no cleavage-specificity. Carbamidomethylation (+57.02146 C) was considered a static modification. Deamidation in presence of H218O (+2.988261 N), GlcNAc (+203.079373 N), oxidation85DM2\301045646.1PATENT Docket No. Y7969-99073 (+15.994915 M) and N-terminal pyroglutamate formation (-17.026549 Q) were considered differential modifications. Data were searched with 50 ppm precursor ion tolerance and 50 ppm fragment ion tolerance. Identified proteins were filtered using DTASelect2 (67) and utilizing a target-decoy database search strategy to limit the false discovery rate to 1%, at the spectrum level (68). A minimum of 1 peptide per protein and no tryptic end per peptide were required and precursor delta mass cut-off was fixed at 15 ppm. Statistical models for peptide mass modification (modstat) were applied. Census2 (69) label-free analysis was performed based on the precursor peak area, with a 15 ppm precursor mass tolerance and 0.1 min retention time tolerance. “Match between runs” was used to find missing peptides between runs. Data analysis using GlycoMSQuant (39) was implemented to automate the analysis. GlycoMSQuant summed precursor peak areas across replicates, discarded peptides without NXS, discarded misidentified peptides when N-glycan remnant-mass modifications were localized to non-NXS asparagines and corrected / fixed N-glycan mislocalization where appropriate.

[0416] Animals and immunizations

[0417] Twenty-four healthy adult Indian-origin rhesus macaques (Macaca mulatta) were used in this study. The animals were housed at the Emory National Primate Research Center (ENPRC). All procedures were approved by the Emory University Institutional Animal Care and Use Committee (IACUC) under protocol 202000087. Animal care facilities are accredited by the U. S. Department of Agriculture (USDA) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.

[0418] Four groups of six RMs were immunized at weeks 0 and 8. In each group All immunization were split between two limbs in the deltoid area. One group received subcutaneous immunizations 100 ug of ApexGT6 soluble trimer protein (ApexGT6 congly gpl40) and 750 ug of SMNP adjuvant. Another group received intramuscular (IM) immunizations of 100 pg of mRNA-LNP encoding cleavage-independent membrane-bound ApexGT6 (ApexGT6 L14 gpl51). A third group was immunized IM with 100 ug of soluble trimer BG505 MD39.3 and 750 ug of SMNP. The final group received lOOug of membrane-bound BG505 MD39.3 gp 151 mRNA-LNP via IM injections.

[0419] Tissue collection and processing.

[0420] Blood was collected throughout the study in NaCitrate CPT tubes for peripheral blood mononuclear cells (PBMCs) and plasma isolation and frozen. Serum was collected via serum clot86DM2\301045646.1PATENT Docket No. Y7969-99073 tubes and frozen. Lymph node fine needle aspirates were collected, processed, and frozen as previously described (46).

[0421] Analysis of plasma by ELISA

[0422] 96-well plates were coated overnight at 4°C with 6x-His (SEQ ID NO: 18) Epitope Tag Rabbit Antibody (Genscript, Cat. No. A00174) at 2ug / mL in PBS. Plates were washed 3 times with PBS, 0.2% Tween (PBS-T), and blocked with 5% milk and 1%FBS in PBS-T for Ih. Subsequently, 2ug / mL of the purified His-tagged trimers was added for 2h in PBS, after which the plates were washed three times with PBS-T. Serial dilutions (3-fold factor) of serum (1:100 dilution) in 1% FBS PBS-T were added to the plates for 1 h, after which the plates were washed again three times with PBS-T before the addition of anti-human conjugated peroxidase at 1:5000 for 1 h. After four final washes, binding was detected by the addition of TMB substrate and measured by absorbance at 450 and 570nm. Background subtraction was performed by subtracting the 570 nm value from the corresponding 450 nm value. Data were subsequently analyzed in Prism (Prism vl0.2; GraphPad Software).

[0423] Negative stain Electron Microscopy Polyclonal Epitope Mapping (nsEMPEM)

[0424] After heat inactivation at 56°C for 1 hr, IgG was purified from plasma samples on an AKTAPure Protein Purification System (Cytiva) on a HiTrap MabSelect PrismA column (Cytiva). Purified IgG was then digested into Fab using liquid papain as described in the EMPEM protocol (70). Once isolated, 750 pg of Fab was complexed with 15 pg of ApexGT6 trimers and left at RT overnight. The following day, trimer-Fab complexes were purified and isolated from unbound Fabs via SEC (Superdex Increase 200; Cytiva). Samples were then concentrated using 10 kDA Amicon® concentrators (Millipore).

[0425] Carbon-coated Cu-400 mesh grids (Electron Microscopy Sciences) and Nano-W™(Nanoprobes) stain were using for negative stain sample grid preparation. The grids were glow discharged with a PELCO easiGLOW (Ted Pella Inc) before sample application. Immediately before sample application, samples were diluted to 0.02 pg / pL in TBS (50 mM Tris pH 7.4, 150 mM NaCl) before 3 pL was applied to the grid. After blotting off the sample, 3 pL of Nano-W™ stain was applied, held for 7 seconds, blotted and waited for 10 seconds, another 3 pL of Nano-W™ stain was applied and held for 15 seconds before a final blot.

[0426] Image collection was done on an FEI Talos microscope (Thermo Fisher Scientific) at 73,000x magnification and 1.98-pixel size using Leginon (71) and initial data processing was 87DM2\301045646.1PATENT Docket No. Y7969-99073 conducted through Appion (72). 2D and 3D Classification and 3D refinement was done through Relion v3.0(73). EM maps were visually presented using UCSF Chimera (74) and Segger (75) was used for segmentation.

[0427] Cryo-EM sample preparation

[0428] For RM038, 200 ug ApexGT6 trimer was complexed overnight at RT with 300 ug of Fabs of RM038 and RM20A3. The sample was then purified via SEC (Superdex 200 Increase; Cytiva) to isolate the complex and remove excess, unbound Fabs. For RM018, 200 [tg of Apex GT6 trimer was complexed overnight with 136.4 pg of RM20A3 Fab at RT and then the following day, 68.2 pg IgG of mAb 018 was added to the sample and allowed to complex with ApexGT6 and RM20A3 for 30 minutes. Samples were then concentrated to 4-5 mg / ml. A Vitrobot Mark IV (Thermo Fisher Scientific) was used to vitrify the samples with the chamber temperature set to 10°C and humidity at 100%. Quanitifoil Cu 1.2 / 1.3 300C-mesh (Quanitfoil Micro Tools GmbH) were glow discharged for 25 s at 15 mA by a PELCO easiGLOW (Ted Pella Inc) prior to sample application. Before samples were applied to the glow discharged grids, lauryl maltose neopentyl glycol (LMNG) was added to a final concentration of 0.005 mM. Blot times varied from 5-7 seconds before plunge freezing into liquid ethane, previously cooled by liquid nitrogen.

[0429] Cryo-EM data collection

[0430] Samples were collected on a 200 kV Glacios (Thermo Fisher Scientific) with an autoloader and mounted Falcon 4 detector (Thermo Fisher Scientific). EPU software (Thermo Fisher) was used for automated data collection. Micrographs were collected at a magnification of 190,000x with a resulting pixel size of 0.725.

[0431] Cryo-EM data processing

[0432] CryoSPARC (76) Live was used for preprocessing of the micrographs and further processing was done in CryoSPARC 3.2 and 4.4. For the monoclonals, particles were picked using blob and template picker before being iteratively 2D classified to remove junk particles. Once good particle stacks were obtained, low resolution, initial 3D volumes were generated using Ab Initio in CryoSPARC. These initial maps were then used to refine the volumes using particle information to generated high-resolution maps after multiple rounds of refinements (homogenous, non-uniform, and local).

[0433] Model building and figure preparation88DM2\301045646.1PATENT Docket No. Y7969-99073

[0434] Initial models for Apex GT6 and RM20A3 and RM038 were generated using UCSF Chimera Modeller (77) using PDB: 7T74. For RM018 Fv, initial model was generated through SAbPred (75) ABodyBuilder-ML (79) (VH framework: 5il8; VL framework: 7tp4; VH-VL orientation: 5n4j). Iterative rounds of model building, and refinements were done through Coot vO.9.8.8 (80), Phenix vl.20.1 (57), and Rosetta (52). Structures were validated using Molprobity (53) and EMRinger (84). Preparations for figures were done through ChimeraX (55).

[0435] Flow cytometry and sorting

[0436] ApexGT6 and ApexGT6. KO probes were prepared by mixing with fluorophore-conjugated streptavidin in lx PBS at room temperature in 1 / 3 increments over 45 minutes. FNA or PBMC samples were thawed and recovered in fully supplemented RPMI (10% (v / v) FBS, Ix pen / strep, 1xGlutaMAX). Live cells were counted and stained with ApexGT6. KO and ApexGT6 probes sequentially for 20 minutes each at 4°C, followed by staining with an antibody master mix and individual anti-human hashtag antibodies at a concentration of 2.5 pg per 5 million cells for 30 minutes at 4°C. PBS with 2% (v / v) FBS was used as FACS buffer. Samples were sorted on a FACSymphony S6 (BD Biosciences). Due to dim staining by our CD71 clone, we chose to sort all CD20+CD38‘ ApexGT6++LN B cells to maximize the number of sequences obtained. This population should contain both GC and memory B cells. For sorting Indexed V(D)J, Feature Barcode, and GEX libraries of sorted LN FNA samples were prepared according to the protocol for Single Indexed 10X Genomics V(D)J 5' v.1.1, with Feature barcoding kit (10X Genomics). For sorted PBMC samples, Indexed V(D)J, Feature Barcode and GEX libraries were prepared using the Dual Indexed 10X Genomics V(D)J 5’ v.2 with Feature barcoding kit (10X Genomics). Custom primers were designed to target RM BCR constant regions. Primer set for PCR 1: forward, AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC TC (SEQ ID NO: 7); reverse, AGGGCACAGCCACATCCT (SEQ ID NO: 8), TTGGTGTTGCTGGGCTT (SEQ ID NO: 9), TGACGTCCTTGGAAGCCA (SEQ ID NO: 10), TGTGGGACTTCCACTGGT (SEQ ID NO: 19), TGACTTCGCAGGCATAGA (SEQ ID NO: 20). Primer set for PCR 2: forward, AATGATACGGCGACCACCGAGATCT (SEQ ID NO: 21); reverse, TCACGTTGAGTGGCTCCT (SEQ ID NO: 22), AGCCCTGAGGACTGTAGGA (SEQ ID NO: 23), AACGGCCACTTCGTTTGT (SEQ ID NO: 24), ATCTGCCTTCCAGGCCA (SEQ ID NO: 25), ACCTTCCACTTTACGCT (SEQ ID NO: 26). Forward primers were used at a final concentration of 1 pM and reverse primers at 0.5 pM, each per 100 pl of PCR reaction. Libraries89DM2\301045646.1PATENT Docket No. Y7969-99073 were pooled and sequenced on a NovaSeq Sequencer (Illumina). A wetting failure occurred during the 1 Ox Genomics library preparation for the week 10 protein PBMC samples, associated with cell health. Additionally, limited cell availability for three animals precluded further attempts. Consequently, no sequence data were obtained from these samples. Overall, sequence recovery from our PBMC samples was lower than expected, likely due to suboptimal cell health.

[0437] BCR sequencing and processing

[0438] Full-length V(D)J reads were assembled with CellRanger v.3.0, utilizing a custom Rhesus Macaque germline VDJ library (46, 47, 86) and adjusting the constants.py file for a maximum CDR3 length of 110 nucleotides. Gene expression counts were obtained from gene expression libraries using CellRanger v.6, aligning to the Ensemble MmullO genome and incorporating mitochondrial genes from Mmul9. Sequencing data were demultiplexed using the MULTIseqDemux command in Seurat v.4. For B cells with kappa and lambda light-chain contigs, lambda was assigned when both were present. The VDJ sequences were then parsed into an AIRR format using the Change-O pipeline from the Immcantation portal for comprehensive analysis.

[0439] Bioinformatic analysis of sequences

[0440] Sequence Processing

[0441] The output from 10X VDJ contigs was re-annotated using the SADIE 0.4.31 library (github.com / jwillis0720 / sadie) with the "macaque" option as the input annotation species. This resulted in a paired AIRR compliant dataframe (www.frontiersin.org / articles / 10.3389 / fimmu.2018.02206 / full). The dataframe was divided into IGH, IGL, and IGK assigned locus and paired based on 10X hashtag, provided they had exactly one heavy IGH and one IGL or IGK call. Somatic hypermutation was calculated by dividing the number of VH or VK / VL gene mutations by the total length of the VH or VK / VL gene. Inferred germline aa sequences were computed by reverting templated V, D, and J gene segments to their germline sequences, based on the predicted alleles from the antibody nucleotide sequence.

[0442] Apex bnAb-like Definition

[0443] The paired dataframe sequences were defined as Apex bnAb-like if they were >= 24 HCDR3 aa long, using IGHD3-15 with the regular expression “ [DE][DE]Y ".

[0444] Apex bnAb-like (22 aa) Definition90DM2\301045646.1PATENT Docket No. Y7969-99073

[0445] The paired dataframe sequences were defined as Apex bnAb-like (22 aa) if they were > 22 and < 24 HCDR3 aa long, using IGHD3-15 with the regular expression “ [DE][DE]Y. ”.

[0446] Analysis of Apex bnAb-like BCR features was conducted using custom Python functions available in the data repository (github.com / schieflab / ma2024).

[0447] The scripts can be accessed at github.com / schieflab / ma2024

[0448] Cell surface antigenic profiling

[0449] DNA-encoded membrane-bound trimers (Moderna) were transfected into HEK293F suspension cells grown in 293 Freestyle media (Life Technologies) by the 293fectin Transfection Reagent (Gibco) and incubated at 37°C, 125rpm for 2 days. Each antibody solution for antigenic profile test (FIG. 5J) was prepared at 10 ug / mL in FACS buffer (HBSS, 1 mM EDTA, 1% BSA). To note, trimer-specific bnAbs (interface / FP: PGT151, Apex: PGT145, PG9 and PCT64.35S) and non-nAbs (V3: 4025, CD4bs: B6 and F105) were selected to characterize the open vs. closed nature of the membrane-bound trimer; not-trimer-specific bnAbs (N332: PGT121 and CD4bs: 12A12) were selected to evaluate cell surface immunogen expression; RMs mAbs (REtl8_wkl7_023, RJzl8_wkl2_041 and RIol8_wk8_018) were selected to assess binding capacity of ApexGT6 induced Apex bnAb-related precursor mAbs towards the immunogen and Apex epitopes closer to native.

[0450] Cell suspension was distributed onto a deep-well 96-well plate at 1 mL per well and harvested at 500 g for 5 min. Each well of cells was resuspended by 100 uL of 10 ug / mL mAb solution and incubated at 37°C, 125rpm for 1 hr. Cells were washed twice with 150 uL FACS buffer and then stained with SYTOX™ Green Dead Cell Stain (Invitrogen) and Alexa Fluor 647-conjugated anti-human IgG (Jackson Immuno Research) at 37°C, 125rpm for 20 min. Cells were analyzed on a NovoCyte 3000 with NovoSampler Pro FACS sorter (Agilent ACEA) by a BD FACS Diva 6 software (BD Biosciences). Approximately 50k live cells were collected per well. Data were analyzed using FlowJo™ vl0.8 Software.

[0451] Apex bNAb precursor frequency estimates

[0452] Apex bnAb precursor searching and frequency estimates were performed as described previously (Willis et al., 2016), with modifications to JH gene usages of CH01-04. PySpark scripts used in this analysis are available at github.com / SchiefLab / Ma2024 along with instructions on setting up an EMR cluster.91DM2\301045646.1PATENT Docket No. Y7969-99073

[0453] Neutralization assay

[0454] MAb neutralizing activity was assessed using single round of replication in TZM-bl target cells. Briefly, pseudoviruses were generated by co-transfection of HEK-293T cells with an Env-expressing plasmid and an Env-deficient genomic backbone plasmid (pSG3AEnv). Pseudoviruses were harvested 72 h post transfection for use in neutralization assays. Individual pseudovirus stocks were mixed with titrated amount of mAbs and pre-incubated 1 h at 37°C with before addition to target cells in presence of 5 ug / mL DEAE-dextran. Infectivity was assessed after 72 h using Luciferase Assay (Promega) readout reagent kits as per manufacturer instructions. All experiments were carried out in duplicate and repeated at least twice. Neutralization was calculated as the percentage loss of infectivity relative to pseudovirus only control wells. To determine neutralization IC50 (concentration yielding 50% neutralization) values, the dose-response curves were fitted using constrained (min=0, max=100) nonlinear regression in Prism 8 (GraphPad).

[0455] Statistical analysis

[0456] Statistical analyses and graphing were carried out using GraphPad Prism for the experimental data. Mann- Whitney compare ranks test was used to compare two experimental groups, and one-way analysis of variance (ANOVA) with Dunn’s multiple comparison test was used for comparing more than two groups. Details of the statistical test and number of replicates are indicated in the figure legends. A value of *P* < 0.05 was considered statistically significant.

[0457] Sequences and binding affinity data for rhesus macque Apex bnAb-like mAbs

[0458] The sequences of the mAbs produced from selected representative Apex bnAb-like precursors from RMs and the binding affinities as evaluated using SPR are shown in the following Table 5. RMs were immunized with the ApexGT6 congly protein as described in in Example 3 or mRNA-LNP encoding cleavage-independent membrane-bound ApexGT6 (ApexGT6 L14 gp 151) as described in Example 6. Responses are categorized as either a germinal center (GC) or memory B cell response derived from bnAb-precursor B cells (MBC).Table 5. Antibody sequences and binding affinity data obtained via SPRAffinity by SPR Anti Imm GC Heavy SEQ H SEQ Light SEQ LCD SEQ ApexGT6 Ape Apex Ape body uniz or Chain ID C ID Chain ID R3 ID xGT V2b xG Nani ation MB Nos. D Nos. Nos. Nos. 6. K T6- e C O DK92DM2\301045646.1PATENT Docket No. Y7969-99073 R3REt Prot GC QVTLKES 27 T 143 ETVVTQ 130 ML 356 2.929e-09 NA 1 798e- 348 18_ ein GPALVKH R EPSLSVS YM 06 7e- Ape TQLLELT A PAGTVT GS 06 xGT CTFSGFS1 1 LTCGLSS GIL6_lo STPGSGV G GSVSTS LngH GWIRQPP E NYPSWY3_W GKALEW E QQTPGQK4_ LASIYWS Q APRTLIY007 DSKYYNT G SINTRPSSLKNRLT F GVPDRF ISTDTSK Y SGSILGN NQVVLT E KAALTIT MTNMDP D GAQADD VDTATY D ECDYYC YCTRATG Y MLYMGS EEQGFYE D GILLFGG DDYDNY N GTRLTV YIEEDVR Y L FDVWGA Y GVLVTVS T S E E D V R F D V RPb Prot GC EVQLVES 28 A 144 DIVMTQ 137 LQS 357 2.332e-08 NA 3734e- 206 18_ ein GGGLAKP K TPLSLPV IEF 06 Oe- Ape GGSLRLS G TPGEPAS PFT 06 xGT CAASGFT W LSCRSSQ6_lo FSSYPMH G SLLDSDngH WVRQAP G GYTCLD3_W GKGLEW A WYLQRPK4_ VSAINSG G GQSPQLOil GSTYYAD E LIYEVSNSVKGRFT D RVSGVP ISRDNSK Y DRFSGS NTLSLQM Y GSDTDF NSLRAED G TLKISRV TAVYYC Y EAEDVG AKGWGG Y VYYCLQ AGEDYY S SIEFPFTF GYYSFLG F GPGTKL DYNSLDV L DIK WGRGVL G VTVSS D Y N S L DV93DM2\301045646.1PATENT Docket No. Y7969-99073 REt Prot GC EVQLVES 29 A 145 QPVLTQ 152 QT 358 NA NA NA NA 18_ ein GGGLVQP K TPSASAS WTApe GGSLRLS D LGASVK TGxGT CTGSGFT E LTCTLSS VRI6_lo FSSYGMS L GHSNYAngH WVRQAP E IAWHQQ3_W GKGLEWI L EQGKAPK8_ SSINSGG Y RFLMRL012 VGTDYA I NSVGSHDSVKGRF P SKGDGIP TISRDNS K DRFSGSS KNTLSLQ V SGAERY MNNLSSE S LTISNLQ DTAVYY N SEDEAD CAKDELE E YYCQTW LYIPKVS D TTGVRIF NEDDYSS D GGGTRL SKILEGD Y TVL YWGRGV S LVTVSS S S K I L E G D Y REt Prot GC EVQLVES 30 A 146 QPVLTQ 252 QT 359 NA NA NA NA 18_ ein GGGLVQP K SPSASAS WTApe GGSLRLS D LGASVK TGxGT CAASGFT Q LTCTLSS VR6_lo FSSYGMS L GHSNYA VngH WVRQGP E IAWHQQ3_W GKGLDWI L EQGKAPK8_ SSIDSGGL Y RYLMRL013 STDYADS I NSVGSHVKGRFTI P SKGDGIP SRDNSKN K DRFSGSS TLSLQMN V SGAERY SLRAEDT S LTISNLQ AVYYCA N SEDEAD KDQLELY E YYCQTW1PKVSNE D TTGVRVDDYSSYN D FGGGTR TLDGDY Y LTVL WGRGVL S VTVSS S Y N T L D G DY94DM2\301045646.1PATENT Docket No. Y7969-99073 Riol Prot GC QVQLVQS 31 A 147 DIQMTQ 253 QU 360 1.175e-09 NA NA 4.80 8_A ein GAEVKKP R SPSSLSA SY 8e- pex GASVKLS G SVGDRV GSP 07 GT6 CKASGYT P TITCRAS LTIon FTVHAIS D ENVNNCgH3 WVRQAP V LHWYQ_W GQGLDW V QKPGKAK8_ MGGIIPL A PKLLIYA014 VGMTNY D AFTLQNAQRFQGR Y GVPSRFS VTITADT G GSGSGT STTTAYM D DFTLTIT ELSSLRSE D SLQPED DTAVYY Y VATYYC CARGPDV G QHSYGS VADYGD Y PLTFGG DYGYHY H GTKVEI RPEDNYL Y K DIWGPGT R PITISS P E D N Y L D IRiol Prot GC QVLLVQS 32 A 148 DIQMTQ 254 QH 361 3.184e-09 NA NA 3.27 8_A ein GADMKN R SPSSLSA SY 3e- pex PGASVKL G SVGDRV GA 07 GT6 SCEASGY P TITCRAS PLTIon TFSIHAIS D ENVNNYgH3 WVRQAP V LHWYQ_W GQGLEW V QKPGKAK8_ MGGIIPL A PKLLIYA015 VGITNYA D ASTLQSQKFQGR Y GVPSRFT VTITADT G GSGSGT STSITYM D DFTLTIS ELSSLRSE D SLQPED DTAMYY Y VATYYC CARGPDV G QHSYGA VADYGD Y PLTFGG DYGYYY Y GTKVEI RPENLYL Y K DIWGPGT R PITISS P E N L Y L D IRiol Prot GC QVQLLQS 33 A 149 QAALTQ 255 SSY 362 4.054e-07 NA NA NA 8_A cin GAEVKKP R PPSVSGS ADpex GASVKLS G PGQSVTI SNTGT6 CKASGYT G SCTGTSS LVJon FSIYAISW G DIGGYN95DM2\301045646.1PATENT Docket No. Y7969-99073 gII3 VRQAPG T YVSWYL_W QGLEWM Y QHPGKAK8_ GGIIPRVG G PKLMIY016 ITTYAQK D DVSKRPFQGRVTI E SGVSDR TADTSTS Y FSGSKSA TAYMELS G NTASLTI SLRSEDT D SGLQAE AVYYCA F DEADYY RGGGTY Y CSSYAD GDEYGDF T SNTLVF YTDGQY D GGGTRL YYDRTY G TVL HFDYWG Q QGVLVT Y VSS Y Y D R T Y H F D YRiol Prot GC QVQLVQS 34 S 150 QAALTQ 256 SSY 362 1.616e-10 NA 2202e- 1 31 8_A ein GAEVKKP R PRSVSES AD 06 le- pex GASVKLS G PGQSVT SNT 08 GT6 CKASGYT L FSCTGTS LVIon FSIYAITW G SDIGGYgH3 VRQAPG P NYVSWF_W QGLEWM S QQHPETK8_ GG1IPLVG 1 APKLM1018 ITNYAQK E YEVSKRFQGRVTI T PSGVSD TADTSTT E RFSGSKS TAYMELS D GNTASL SLRSEDT D TISGLQA AVYYCSR Y EDEADY GLGPSIET D YCSSYA EDDYDYS Y DSNTLV YTSEYNL S FGGGTR LHVWGR Y LTVL GVLVTVS T S S E Y N L L H VRiol Prot GC DVQLVES 35 A 151 DIVMTQ 257 MQ 363 3.746e-08 NA NA 8.88 8_A ein GGGLVQP R TPLSLPV TLQ Oe- pex GGSPRLS M TPEEPAS TPF 07 GT6 CGASGFT G ISCRSSQ TIon FTSHGMH R SLLDSDgH3 WVRQAP R GYTHLHW GKGLEW S WYLQKP96DM2\301045646.1PATENT Docket No. Y7969-99073 K8_ VAVISSD Y GQSPQL024 GSKKNY D LIYLVSSADSVKD D RASGVP RFTISRD D DRFSGS NSKNML Y GSGTDF YLQMNT D TLKINRV LKLEDTA H EAEDVG VYYCAR Y VYYCM MGRRSY Y QTLQTPF DDDYDH F TFGPGT YYFAGDP A KLDIK PLLDVW G GRGLLVT D VSS P P L L D VRiol Prot GC DVQLVES 36 D 36 QAALTQ 258 SSY 364 2.678e-06 NA NA NA 8_A ein GGGLVQP V PRSVSGS AGpex GGSLRLS Q PGQSVTI SNTGT6 CGASGFT L SCTGTSS FYIIon FSSHGMH V DIGGYNgH3 WVRQAP E YVSWYQ_W GKGLEW S QHPGTAK8_ VAVISSD G PKLMIY025 GVKKNY G EVSKRPSADSVKD G GVSDRF RFTISRD L SGSKSG NSKNML V NTASLTI YLQMNN Q SGLQAE LKLEDTA P DEADYY VYYCAR G CSSYAG MGRRSN G SNTFYIF DDDYEY S GAGTRL YYFAGDS L TVL PLLDVW R GRGLLVT L VSS S C G A S G F T F S S H G M H W V R QAP97DM2\301045646.1PATENT Docket No. Y7969-99073 G K G L E W V A V I S S D G V K K N Y A D S V K D R F T I S R D N S K N M L Y L Q M N N L K L E D T A V Y Y C A R M GRR98DM2\301045646.1PATENT Docket No. Y7969-99073 S N D D D Y E Y Y Y F A G D S P L L D V W G R G L L V T V S SRiol Prot GC EVQLVES 37 A 153 NIVMTQ 259 MQ 365 6.116e-09 NA NA 5.54 8_A ein GGGLVQP K 1PLSLPV TIE 9e- pex GGSLRLS D TPGEPAS FP 08 GT6 CAASGFT T ISCRSSQ WTIon FSNYGM R SLLDSDgH3 HWVRQA Y GYICLD_W PGKGLE Y WYLQKPK8_ WVAVISY E GQSPQL026 DGSQKY D LIYEVSNYEDSVKD E RVSGVP RFTISRD Y DRFSGS NSKSMLY G GSGTDF LQMNNL Y 1LKISRVKLEDTAV H EAEDVG YYCAKD Y VYYCM TRYYEDE I QTIEFPW YGYHYIF F TFGQGT AGRIYGL A KVEIR DSWGQG G VVVTVSS R I Y G L DS99DM2\301045646.1PATENT Docket No. Y7969-99073 Riol Prot GC EVQLVQS 38 A 154 DIVMTQI 260 MQ 366 7.676e-09 NA 2071e- 793 8_A ein GAEVKRP T PLSLPVT TLQ 06 8e- pex GESLKISC D PGEPAAI TPL 08 GT6 TASGYTF V SCRSSQS TIon TNNWIS N LLDSDGgH3 WVRQLP G YTQLHW_W GEGLEW D YLQKPGK8_ MGAIHPG Y QSPQLLI028 DSDTRYS E YLVSNRPSFQGQV D ASGVPD TISVDKSI D GFSGSGS STAYLQ Y GTDFTL WSSLKAS G KINRVE DSATYYC Y AEDAGV ATDVNG Y YYCMQT DYEDDY Y LQTPLTF GYYYTV T GGGTKV GRGHYF V EIK DFWGQG G VLVTVSS R G H Y F D F RGp Prot GC EVQVAES 39 V 155 DIVMTQ 261 MQ 367 5.285e-10 NA 2951e- 1 54 18_ ein GGGLVQP R TPLSLPV SVE 06 7e- Ape GGSLRLA G TPGEPAS YPF 08 xGT CEASGFT R ISCRSSQ T6 lo FSGYEVH M SLFDGDngH WVRQAP Y SGNTYL3_W GKGLESV Y DWYLQK12 SVIGADN E KPGQSP_035 SYTHYAD D QLLIYMSVKGRFT D LSNRAS ISRDNAK Y GVPDRF NSLSLQM G SGSGSG NSLTAAD Y TDFNLKI TAVYYC Y SRVEVE VRGRMY A DVGVYY YEDDYG T CMQSVE YYATGD G YPFTFGP YNGLDS D GTKLD1WGQGVV Y K VTVSS N G L D S RGp Prot GC EVQLAES 40 V 156 DIVMTQ 262 MQ 368 8.802e-09 NA 6.196e- 5 15 18_ ein GGGLVQP R TPLSLPV GV 06 le- Ape GGSLRLS G TPGEAA EYP 08 xGT CEASGLT R SISCRSS FT6_lo FSAYEVH M QSLFDGngH WVRQAP Y DSANTY3_W GQGLESV Y LDWYLQSVIGADN E KPGQSPSYTHYAD D QLLIYM100DM2\301045646.1PATENT Docket No. Y7969-99073 K12 SVKGRFT D LSNRAS_036 ISRDSGK Y GVPDRFNSLSLQM G SGSGSG NNLRAA Y TDFKLKI DTAVYY Y SRVEVE CVRGRM A DVGIYY YYEDDY T CMQGVE GYYATG G YPFTFGP DYYGLDS D GTKLDI WGQGVV Y K VTVSS Y G L D S RJzl Prot GC QCVEQLV 41 DIVMTQ 263 MQ 369 5.646e-11 NA 7658e- 776 8_A ein ESGGGLV TPLSLPV GIQ 07 5e- pex QPGASLR TPGEPAS LPY 09 GT6 LSCAASE ISCRSSQ SIon FTFSDSD SLLDSEAgH3 MHWVRQ GNTYLD_W VPGQGLE WYLQRPK12 WVSAISL GQSPQL_038 GGDTHYP LIYEVSNDSVKGRF RASGVP HSRDNA DRFSGS KNSLYLQ GSDTDF MNSLRPE TLKVSR DTAVYY VEAEDV CARGGR GVYYC GGYEDD MQGIQL YGYFYFI PYSFGQ GGRRSLD GTKVE1VWGRGA K LVTVSS RJzl Prot GC QCVEQLV 42 DIVMTQ 264 MQ 369 1.024e-10 NA 4893e- 147 8_A ein ESGGGLV TPLSLPV GIQ 06 7e- pex QPGASLR TPGEPAS LPY 08 GT6 LSCAASE ISCRSSQ SIon FTFSNYD SLLDSEAgH3 MHWVRQ GNTYLD_W VPGKGLE WYLQRPK12 WVSAISL GQSPQL_039 GGDTRYP LIYEVSNDSVKGRF RASGVP HSRDNA DRFSGS KNSLYLQ GSDTDF MNSLRPE TLTVSRL DTAVYY EAEDVG CGRGGR VYYCM GGYEDD QGIQLPY YGYLYIV SFGQGT GGRRSLD KVEIK VWGRGV LVTVSS101DM2\301045646.1PATENT Docket No. Y7969-99073 RJzl Prot GC EVQLVES 43 A 157 DIRMTQ 265 LQ 370 7.080e-08 NA 1.103e- 1 83 8_A ein GGGLVQP R SPSSLSA YN 05 Oe- pex GGSLRLS D SAGDRV SKP 06 GT6 CAASGFT G HTCRAS FTIon FSRYGM T QDISICLgH3 HWVRQA A SWFQQK_W PGKGLE Y PGKPPKK12 WVTVISH Y RLIHAAS_040 DGSQND E SLESGVPYGDSVK D SRFSGSG GRFTISR D SGTEFTL DNSKNIL Y HNSLQP YLQMND E EDFATY LKLEDTA C FCLQYN VYYCAR Y SKPFTFG DGTAYY S PGTKLDI EDDYECY F K SFNECNS N LDVWGR E GVLVTVS C S N S L D V RJzl Prot GC EVQLVES 44 A 158 DIQMTQ 266 LQ 370 2.433e-10 NA 1.074e- 103 8_A ein GGGLVQP R SPSSLSA YN 07 2e- pex GGSLRLS D SAGDRV SKP 08 GT6 CAASGFT G HTCRAS FTIon FSRYGVH T QDISIYLgH3 WVRQAP A SWFQQK_W GKGLEW Y PGKAPKK12 VTVISHD Y RL1YTAS_041 GSKNDY E SLESGVPGDSVKD D SRFSGSG RFTISRD D SGTEFTL NSKNILY Y HNSLQP LQMNDL E EDFATY TLEDTAV Y FCLQYN YYCARD Y SKPFTFG GTAYYE S PGTKLDI DDYEYYS F R FNDEYNS N LDVWGR D GVLVTVS E S Y N S L D V RJzl Prot GC EVQLVES 45 A 159 DIQMTQ 267 LQ 370 9.382e-11 NA 7.208e- 4 11 8_A ein GGGLVQP R SPSSLSA YN 06 Oe- pex GGSLRLS D SAGDRV SKP 09 GT6 CAASGFT G HTCRAS FTIon FSRYGM T QDISIYLgH3 HWVRQA A SWFQQR_W PGKGLE Y PGEAPKWVTVISH Y RLIYAAS102DM2\301045646.1PATENT Docket No. Y7969-99073 K12 DGSKND E SLESGVP_042 YGDSVK D SRFSGSGDRFTISR D SGTEFTL DNSKNIL Y UNSLQP YLQMND E EDFATY VKLEDTA Y FCLQYN VYYCAR Y SKPFTFG DGTAYY S PGTKLD EDDYEY F VR YSFNDYS N SLDFWGR D GVLVTVS Y S S S L D F REt Prot GC EVQLVQS 46 A 160 DIVMTQ 268 MQ 371 3.284e-08 NA NA 1.16 18_ ein GGGLVQP K TPLSLPV AL 5e- Ape GGSLRLS D ILGEPAS AFP 06 xGT CGASGFA R ISCKSSQ YS6_lo FSSYGMF S SLLHSEDngH WVRQAP P GNTYLE3_W EKGLEWI Y WYLQKPK12 SAVNSGG R GQSPQV_033 ESTKYAD D LIYELSNSVKGRFT D RASGVP VSRDNSK Y DRFSGS NTLTLQL G GSDTDF NSLRAED Y ILKISRV TAVYYC Y EAEDVG AKDRSPY Y VYYCM RDDYGY A QALAFP YYAGLD G YSFGQG RFDVWG L TKVEIK AGVLITV D SS R F D V REt Prot GC EVQLVQS 47 A 161 DIVMTQ 269 MQ 372 4.357e-10 NA 7221e- 238 18_ ein GGGLVQP K TPLSLPV ALE 07 2e- Ape GGSLRLS D TLGEPAS FPY 08 xGT CAASGFT R ISCKSSQ S6 lo FSSYGMF S SLLHSEDngH WVRQAP P GNTYLE3_W EKGLEWI Y WYLQKPK12 SAINSGG Q GQSPQV_034 DDTRYA D LIYELSNDSVKGRF D RASGVP TVSRDNS Y DRFSGS MNTLSLQ G GSDTDF LNSLRPE Y TLKINRV DTAVYY Y EAEDVG CAKDRSP Y VYYCM YQDDYG L QALEFP YYYTGL G YSFGQG DRFDVW L TKVEIKD103DM2\301045646.1PATENT Docket No. Y7969-99073 GAGVLIT R VSS F D V REt Prot GC EVQLVES 48 A 162 DIVMTQ 270 MQ 373 1.723e-08 NA NA NA 18_ ein GGGLVQP N TPLSLPV ALApe GGSLRLS L TLGESAS VFPxGT CVASGFT A ISCRSSQ WT6_lo FTSYGMY T SLLHSEDngH WVRQAP Y GNTYLE3_W GKGLEWI E WYLQKPK4_ SAINGGG D GQSPQV008 DSTYYAD D LIYEVSNSVKGRFT Y RASGVP ISRDNSK G DRFSGS NTLSLQM Y GSDTDF NSLRAED Y TLKVSR TAVYYC Y VEAEDV ANLATYE T GVYYC DDYGYY S MQALVF YTSINRF I PWTFGQ DVWGAG N GIKVEI VLVTVSS R K F D V REt Prot GC EVQLVES 49 A 163 DIVMTQ 271 MQ 374 1.259e-07 NA NA 388 18_ ein GGGLVQP K TPLSLPV GIE 2e- Ape GGSLRLS D TPGEPAS FPL 07 xGT CAASGFT T ISCRSSQ T6_lo FSSYGMY T SLLDSEDngH WVRQAP Y GNTYLE3_W GKGLEWI F WYLQKPK4_ SAINSGS E GQSPQP009 DRTYYA D LIYEVSNDSVKGRF D RASGVP HSRDNS Y DRFSGS KNTLSLQ A GSDTDF MNSLRAE Y TLKISRV DTAVYY Y EAEDVG CAKDTTY R VYYCM FEDDYAY R QGIEFPL YRRTYNS T TFGGGT LDVWGR Y KVEIK GVLVTVS N S S L D V REt Prot GC EVQLVES 50 A 164 DIVMTQ 272 MQ 374 1.901e-08 NA NA 1.07 18_ ein GGGLVQP K TPLSLPV GIE 7e- Ape GGSLRLS D TPGDPA FPL 07 xGT CEASGFIF T SISCRSS T6_lo SSYGMY T QSLLDSEngH WVRQAP Y DGNTYL3_W GKGLEWI Y EWYLQKSAINSGG E PGQSPQPDNTYYA D LIYEVSN104DM2\301045646.1PATENT Docket No. Y7969-99073 K4_ DSVKGRF D RASGVP010 TISRDNS Y DRFSGSKNTLSLQ V GSDTDF MNSLRAE Y TLKISRV DTAVYY Y EAEDVG CAKDTTY R VYYCM YEDDYV R QGIEFPL YYRRTY T TFGGGT NSLDVW Y KVEIK GRGVLVT N VSS S L D VRiol Prot GC QCVEQLV 51 V 165 DIVMTQ 273 MQ 375 1.128e-10 NA 6.034e- 657 8_A ein ESGGGLV R TPLSLPV AL 05 2e- pex RPGASLR G TPGEPAS QFP 08 GT6 LSCAASE S ISCRSSQ LTIon FTFSDYD E SLLHSDgH3 MHWVRQ A GKTFLC_W APGKGLE Y WYLQKPK8_ WVSGISI E GQSPQL021 GGDTNYP D LIHEVSNDSVKGRF D RASGVP TISRDNA Y DRFSGS KNSLYLQ D GSGTDF MNSLRPE Y TLKISRV DTAVYY Y EAEDVG CVRGSEA Y VYYCM YEDDYD T QALQFP YYYTRHS R LTFGGG LDVWGR H TKVEIK GVLVTVS S S L D VRiol Prot GC QCVEQLV 52 V 166 DIVMTQ 273 MQ 375 1.651e-08 NA NA NA 8_A ein ESGGGLV R TPLSLPV ALpex QPGASLR G TPGEPAS QFPGT6 LSCAASE S ISCRSSQ LTIon FTFSNYD E SLLHSDgH3 MHWVRQ A GKTFLC_W APGKGLE F WYLQKPK8_ WVSGISV E GQSPQL022 GGDTNYP D LIHEVSNDSVKGRF D RASGVP TISRDNA Y DRFSGS KNSLYLQ D GSGTDF MNSLRAE Y TLKISRV DTAVYY Y EAEDVG CVRGSEA N VYYCM FEDDYDY T QALQFP YNTRHSL R LTFGGG DVWGRG H TKVEIK VLVTVSS S L DV105DM2\301045646.1PATENT Docket No. Y7969-99073 RJzl Prot GC EVQLVES 53 A 167 DIVMTQ 274 MQ 376 9.181e-10 NA 3.715e- 1 89 8_A ein GGGLVQP K TPLSLPV GIQ 05 6e- pex GGSLRLS D TPGEPAS LPL 06 GT6 CAASGFT R ISCRSSQ TIon FSNYGM Q SLLKSEEgH3 YWVRQA E GNTYLD_W PGKGLE S WYLQKPK12 WISTINSG Y GQSPQL_043 GDNTEH E LIYEVSNADSVKG E RASGVP RFTVSRD D DRFSGS NSKNTVS Y GSDTDF LQMNSLR D TLKISRV PEDTAVY N EAEDVG YCAKDR F IYYCMQ QESYEED Y GIQLPLT YDNFYPG P FGGGTK ASLIWGQ G VEIK GVLVTVS A S S L I RJzl Prot GC EVQLVES 54 A 168 DIVMTQ 274 MQ 376 2.690e-09 NA NA 1.18 8_A ein GGGLVQP K TPLSLPV GIQ 8e- pex GGSLRLS D TPGEPAS LPL 06 GT6 CETSGFT R ISCRSSQ TIon FSSYGMY Q SLLKSEEgH3 WVRQAP E GNTYLD_W GKGLEWI S WYLQKPK12 SAINSGN Y GQSPQL_044 NNTDHA E LIYEVSNESVKGRF E RASGVP TISRDNS D DRFSGS KNTLSLQ Y GSDTDF MNSLRPE D TLKISRV DTAVYY Y EAEDVG CAKDRQ F IYYCMQ ESYEEDY Y GIQLPLT DYFYPGA P FGGGTK SLIWGQG G VEIK VLVTVSS A SL1RJzl Prot GC EVQLVES 55 A 168 DIVMTQ 275 MQ 377 4.970e-09 NA 3.151e- 169 8_A ein GGGLVQP K TPLSLPV AIQ 05 Oe- pex GGSLRLS D TPGEPAS LPL 05 GT6 CAASGFT R ISCRSSQ TIon FSSYGMY Q SLLKSEEgH3 WVRQAP E GDTYLD_W GKGLEWI S WYLQKPK12 SAINSGG Y GQSPQL045 DNTDHA E LIYEVSNDSVKGRF E RASGVP TISRDNS D DRFSGS KNTLSM Y GSDTDF QMNSLRP D TLKISRV EDTAVY Y EAEDVGYCAKDR F IYYCMQ106DM2\301045646.1PATENT Docket No. Y7969-99073 QESYEED Y AIQLPLT YDYFYPG P FGGGTK ASLIWGQ G VEIK GVLVTVS A S S L I RJzl Prot GC QCVGQL 56 DIVMTQI 276 MQ 378 6.342e-08 NA 1.933e- NA 8_A ein VESGGGL PLSLPVT GV 06 pex VQPGASL PGEPASI QLPGT6 RLSCAAS SCRSSQS LTIon EFTFSTY LLDSENgH3 DMHWVR GNTYLD_W QAPGKGL WYLQRPK8_ EWVSAISI GQSPQL030 GGDTHYP LIYEVSNDSVKGRF RASGVP TISRDNT DRFSGS KNSLYLQ GSDTDF MNSLRAE TLKISRV DTAVYY EAEDVG CRRGRRY VYYCM YEDDYD QGVQLP YYHTED LTFGGG YGLDSW TKVEIK GQGVVV TVSS RAu mR MB QVQLVQS 57 A 169 DIQMTQ 277 QQ 379 8.268E- NA NA 1 94 18_ NA C GAEVKKP R SPSSLSA YN 10 4E- Ape GASVKLS L SVGDRV GA 06 xGT CEASGYT G TITCRAS PLT6_w FSIYAIG G QGISSYLkl0_ WVRKAP L AWYQQ001 GQGLEW Y KPGKAPMGGIIPL Y KPLIYYA VGTTNY E SNLESG AQKFQG D VPSRFSG RVTITAD D SGSGTFFTSTTTAY Y TLTISSL MELSSLT D QPEDFA SEDTAVY Y TYYCQQ YCARLG S YNGAPL GLYYED S TFGGGT DYDYSST T KVEIK DLADNRF D DVWGPG L VLVTVSS A D N R F D V RAu mR MB EVQLVES 58 A 170 DIVMTQ 278 MQ 380 4.925E- NA NA 706 18_ NA C GGGLAQP R TPLSLPV GIQ 09 6E- Ape GGSLRLS M TPGEPAS FPF 09 xGT CAASGFS V ISCRASQ S6_w FSRHGM R SLLNSED107DM2\301045646.1PATENT Docket No. Y7969-99073 kl0_ IIWVRQA A GNTYLD002 PGKGLD A WYLQKPWVSIISFD D GQSPQL GSKEYYA E LIYEVSN DSVKDRF D RASGVP TISRDNS D DRFSGS KNMLYL Y GSDTDF QMNNLK G TLKISRV LEDTAVY Y EAEDVG YCARMV Y VYYCM RAADED Y QGIQFPF DYGYYY T SFGPGT TLRLGAH L KLDIK NSLDVW R GRGILVT L VSS G A H N S L D V RGu mR MB EVQLVES 59 A 171 EIVLTQS 279 QQ 381 1.212E- NA 9.207E- 1.00 18_ NA C GGGLVQP K PATLSLS YS 09 06 4E- Ape GGSLRLS D PGERAT KW 07 xGT CAASGFT R LSCRAS PLT6_w FSINAMY Y QSVSGSkl0_ WIRQAPG Y LAWHQ003 KGLEWIS E QKPGQAAISSGGDI D PRLLIYG TYYADSV D ASSRAT KGRFTIS Y GIPDRFSRDNSKNT G GSGSGT LSLQMNS Y DFTLTIS LRAEDTA Y SLEPEDF VYYCAK Y AVYYCQ DRYYED T QYSKWP DYGYYY E LTFGGG TEPESHN P TKVEIK SLDVWG E RGVLVTV S SS H N S L D V RGu mR MB QVQLVQS 60 A 172 QSVLTQ 280 AA 382 1.467E- NA 2.375E- 2.81 18_ NA C GAEVKKP R PPSASEA WD 10 06 2E- Ape GASVKLS G ARKSVTI DSL 06 xGT CKASGYT H SCSGSSS SG6_w FTSYSVN F NIGSNSV YIkl0_ WVRQAP P SWYQQL004 GQGLEW A PETAPKLMGWINP C LIYYND NNGNTG Y RRASGV YAQKFQ E SDRFSGSGRVTMT D KSGTSA108DM2\301045646.1PATENT Docket No. Y7969-99073 RDTSTST D SLAISGL AYMELSS Y QTEDEA LRSEDTA D DYYCAA VFYCARG Y WDDSLS HFPACYE I GYIFGA DDYDYIC C GTRLTV THDVPGV T L DVWGPG H VLVTVSS D V P G V D V RGu mR MB EVQLVES 61 T 173 QPVLTQ 281 QT 383 1.024E- NA NA 1 91 18_ NA C GGGVVQ R SPSASAS WT 08 4E- Ape PGGSLRL D LGASVK TGI 06 xGT SCAASGF R LTCTLSS VL6_w TFDDYA A GHSSYAIkl0_ MHWVRQ F AWHQQ005 APGKGLE T QQGKAPGISRISW G RYLMRL NSGSISY S NSVGSH ADSVKG Y NKGDGI RFTISRD Y PDRFSGS NAKNSLF E SSGAER LQMDRL D YLTISNL TVEDTAV D QSEDEA YYCTRDR Y DYYCQT AFTGSYY G WTTGIV EDDYGYS Y LFGGGT YTDEGG S RLTVL GLDSWG Y QGVVVT T VSS D E G G G L D S RGu mR MB EVQLVES 62 A 174 DIQMTQ 282 LQF 384 1.843E- NA 8.575E- 419 18_ NA C GGGLAKP G SPSSLSA NG 10 06 8E- Ape GGSLRLS E SAGDTV HP 08 xGT CEASGFT T HTCRAS YS6_w FSRYWM M QGISTFLkl0_ HWVRQA R NWYQQ006 PGKGLE P KPGKAPWISVINS R KRLIYA DGSNTYY Y ASSLESG ADSVQG E VPSRFSG RFTISREN D SGSGTD AKTTLYL D FTLTISS QMDSLRP Y LQPEDF EDTALYY G ATYYCL CAGEIM Y QFNGHPRPRYEDD Y109DM2\301045646.1PATENT Docket No. Y7969-99073 YGYYYT Y YSFGQG DLVDYY T TKVEIK GLESWG D QGVVVT L VSS V D Y Y G L E S RPb mR MB EVQLVES 63 A 175 DIVMTQ 283 MQ 385 1.571E- NA 2.795E- 000 19_ NA C GGGLAKP K TPLSLPV ALE 10 06 000 Ape GGSLRLS Q TLGEPAS FPL 878 xGT CAASGFT S ISCRSSQ T6_w FSRYAM p SLLDSENkl0_ HWVRQA G GNTYLE007 PGKGLE A WYLQKPWVSGMN D GQSPQV SGGSTYY Y LIYEVSN ADAVKG E RASGVP RFTISRED D DRFSGSNSKNTLS E GSDTDF LEMNSLR Y TLKISRV TEDTAVY V EAEDVG YCAKQSP Y VYYCM GADYED Y QALEFPL EYVYYD D TFGGGT TRYGLDS T KVEIK WGQGAV R VTVSS Y G L D S RPb mR MB EVQLVES 64 A 176 EIVVTQS 284 LQR 386 4.055E- NA NA 894 19_ NA C GGGLAKP K PATLSLS SN 09 3E- Ape GGSLRLS D PGERAT WP 08 xGT CAASGFT T LSCRTSQ LT6_w FSGYGM Y SVSNSLkl0_ HWVRQA Y AWYQQ008 PGKGLE E KPGRAPWVSAISG D RLLIYGV GGTTWY D STRATGI ADSVKG Y PDRFSGS RFAISRD G GSGTDF NPKNTLS Y SLTISSL LQMNSLR Y EPEDVG AEDTAM Y VYYCLQ YYCAKD T RSNWPL TYYEDD D TFGGGT YGYYYT D KVEIK DDSTTYY S FDYWGQ T GVLVTVS T S YYF110DM2\301045646.1PATENT Docket No. Y7969-99073 D Y RPb mR MB QVQLVQS 65 A 177 DIVMTQ 285 MQ 387 2.465E- NA 9.385E- 1.05 19_ NA C GAEVKKP R TPVSLPV SLE 10 06 1E- Ape GASVKLS G TLGEAA FP 06 xGE CKASGYS K S1SCRSS WE6_w FSNYAIF G QSLLHSEklO_ WVRQAP G DGNEYL009 GQGLEW A EWYLQKMGGIVPL A PGQSPH VPTTNYA Y LLIYEVS QKFQGR E NRASGV VTTTADT D PDRFSGS STDTVY D GSDEDF MELTSLR Y ELKISRV SEDTAVY G EAEDVG YCARGK Y VYYCM GGAAYE Y QSLEFP DDYGYY Y WEFGQG YTSIRHG E EKVEIK LDSWGQ S GVVVTVS I S R H G L D S RPb mR MB QVQLVQS 66 A 178 QVILTQS 286 QE 388 1.171E- NA NA 5.70 19_ NA C GAEVKKP R PATLSLS YSS 09 4E- Ape GASVKLP H PGERAT SPL 07 xGT CKASGY G LSCRAS E6_w NFNIYAIS A QSVSSSLkl0_ WVRQAP P GWYQQ010 GQGLEW S KPGQAPMGGIIPL E RLLIYGA VGITTYA I SSRAEGI QKFQGR D PDRFSGS VHTADT Y GSGEEFE STSEGYM E LEISSLE ELSSLRSE D PEDFAV DTAIYYC D YYCQEY ARHGAPS Y SSSPLEF TIDYEDD A GGGEKV YAYYNT Y EIK GYNWFD Y VWGPGA N LVTVSS E G Y N W F D V RPb mR MB QVQLVQS 67 A 179 DIQMSQ 287 QQ 389 2.882E- NA 0.00001 NA 19_ NA C GAEVKKP R SPSSLSA GN 09 115Ape GASVKLS L SAGDRV111DM2\301045646.1PATENT Docket No. Y7969-99073 xGT CKASGYT G TITCRAS SNP6_w FSIYAMS G QGISNYL LTkl0_ WVRQAP P NWYQQOil GQGLEW P EPGKAPMGGIIPL S KLLIYN VGITNYA V VNNLAS QKFQGR V GVPSRFS VHTADT D GSGSGT STNTAY Y DFTLTIS MELSSLR E SLQPEDF SEDTAVY D ATYYCQ YCARLG D QGNSNP GPPSVVD Y LTFGGG YEDDYA A TKVEFK YSDTKGH Y GLDSWG S QGVVVT D VSS T K G H G L D S RPb mR MB QVTLKES 68 A 180 QVILTQS 288 YH 390 4.309E- NA NA 5.31 19_ NA C GPALVKP R PATLSLS HSS 10 2E- Ape TQTLTLT L PGERAT GD 08 xGT CTFSGFS N LSCRAS FYS6_w LTISGVG Y QSLSSSLklO_ VGWIRQP E AWYQQ012 PGKALE D KPGQAPWLALIY D RLLIHSA WDDGKH Y SSRATGI YSTSLKS D PDRFSGS RLTISKD Y GSGTEFT TSKNQVV Y LTISSLD LRMTYM S PEDVGV DPVDTAT T YHCYHH YFCARLN E SSGDFYS YEDDYD G FGQGTK YYSTEGG G VEIK SYMQGT S NDAFDF Y WGQGLR M VTVSS Q G T N D A F D F RPb mR MB QVQLVQS 69 A 181 QAALTQ 289 SSY 391 4.394E- NA NA 1.40 19_ NA C GAEVKKP R PPSVSGS AD 09 5E- Ape GASVKLS G PGQSVTI SNT 06 xGT CKASGYT R SCTGTSS LL6_w FSIYAISW P DIGGYN112DM2\301045646.1PATENT Docket No. Y7969-99073 klO_ VRQAPG D YVSWYQ013 QGLEWM P QHPGKAGGIIPLVG T PKLMIY ITNYAQR S DVSERPS FQGRVTI F GVSDRF TADTSTS S SGSKSG TAYMEM E NTASLTI SSLRSED D SGLQAE TAVYLCA D DEADYY RGRPDPT Y CSSYAD SFSEDDY G SNTLLFG GDYYTG D GGTRLT GRGDWY Y VL FDIWGPG Y TPITISS T G G R G D W Y F D I RYu mR MB EVQLVET 70 A 182 DIVMTQ 290 VQ 392 2.676E- NA 2.912E- 4.96 18_ NA C GGGLVQP K TPLSLPI VIA 11 07 9E- Ape GGSLRLS D TPGEPAS FPY 09 xGT CAASGFT F ISCRSSQ S6_w FSSHTMH E SLLHSNkl0_ WVRQAP R GNTHLH014 GKGLEWI T WYLQKPSAINSGG Y GQSPQLGKTYYE Y LIYGGSN DSVKGRF E RASGVP TISRDNS D DRFSGS KNTLSLQ D GSGTDF MNSLRPE Y ILKISKV DTAVYY G EAEDVG CAKDFER D VYYCVQ TYYEDD Y VIAFPYS YGDYYTF Y FGQGTK VEDYYG T VEIK LDSWGQ F GVVVTVS V S E D Y Y G L D S RJrl mR MB QVKLQQ 71 A 183 EIVLTQS 291 HQ 393 9.402E- NA NA NA 8_A NA C WGEGLV R PAILSLS YN 09pex KPSETLS T PGETAT DLLGT6 LTCAVYG P LSCRASE WT_wk GSISGYY L SVGSYLYWNWIR R AWYQQ113DM2\301045646.1PATENT Docket No. Y7969-99073 10_0 QAPGKGL Y KPGQAP15 EWIGYIS Y RLLVHSGNSVITN E ANFRAT YNPSLKS D GIPDRFS RVTISTD D GSGSRT TSKNQFS Y EFTLTIS LRLSSVT D SLEPEDV AADTAV D GVYHCH YYCARTP I QYNDLL LRYYEDD M WTFGQG YDDIMV V TKVEIK DRDAFDF D WGQGLR R VTVSS D A F D F RJrl mR MB EVQLEES 72 A 184 DIVMTQ 292 MQ 394 5.38E-09 NA NA 6.70 8_A NA C GGGLARP K TPLSLPV ALE 5E- pex GGSLRLS F TPGEPAS FPF 07 GT6 CAASGFA R ISCRSSQ T_wk FRSYAM G SLLNSD10_0 HWVRQA E DGNTYL16 PGKGLE Y DWFLQRWVSAISS Y PGQSPQ AGSTKYA E VLIYEVS DSVKGRF D NRASGV TITRDNS D PDRFSGS KNTLSLQ Y GSDTDF MNSLRTE G ILKISRV DTAVYY Y EAEDVG CAKFRGE Y LYYCMQ YYEDDY Y ALEFPFT GYYYSGL S FGGGTK ADSYYW G VEIK GQGVLV L TVSA A D S Y Y RJrl mR MB DVQLVES 73 T 185 DIVMTQ 293 MK 395 7.41E- 10 NA 9.902E- 8.16 8_A NA C GGGLVKP R TPLSLPV ALE 07 5E- pex GGSLRLS G TPGEPAS FPF 06 GT6 CVASGFT G ISCRSSQ T_wk FSSYLMH L SLLNSED10_0 WVRQAP F GNTYLD17 GKGLEW H WYLQKPVSVISESG I GQSPQL GNIYYSD D LIYEVSN SVKGRFT Y RASGVP ISRDNAK D DRFSGS NSLFLQM D GSDTDF NSLRAED D TLEISRV TAVYYCT Y EAEDVG RGGLFHI A VYYCM DYDDDY Y KALEFPFAYYYTG Y114DM2\301045646.1PATENT Docket No. Y7969-99073 RGLDYW Y TFGPGT GQGVLV T KLDIK TVSA G R G L D Y RJrl mR MB QVQLVQS 74 A 186 DIQMTQ 294 QH 396 1.217E- NA NA NA 8_A NA C GAEVKKP R SPSSLSA GY 07pex GASVKVS P SVGDRV GTPGT6 CQASGFT G TITCRAS FT_wk FGNYPTS G FNVNNY10_0 WVRQAP G LNWYQ18 GQGLEW R QKPGKAMGGIVPL F PKLLIYK VGLTNY Y ASTLQS AQKFQG E GVPSRFS RVTITAD D GSGSGT TSTNTAY D DYTFTIS MDLSSLR Y SLQPED SEDMAV D VATYYC YYCARPG Y QHGYGT GGRFYED Y PFTFGPG DYDYYW W TNLDIK GDGDWF G DVWGPG D VPVAVSS G D W F D V RJrl mR MB QVQLQES 75 A 187 QTALTQ 295 YS 397 2.576E- NA 5.198E- 4.43 8_A NA C GPGLVKP R PPSVSKS YG 09 06 1E- pex SETLSLT F LGQSVTI SGS 08 GT6 CTVSGAA P SCTGTS NW_wk IRSYWW R DDVGGY V10_0 NWIRQPP G NDVSWY19 GKGLEWI G QQHPGTGEINGKS E GPRLLIY GSTNYNP D EVSKRPS SLKSRVTI Y GVSDRF SKDASKN Y SGSKSGS QISLRLTS E TASLTIS VIAADTA D GLQAED VYYCARF D EADYYC PRGGEDY Y YSYGSG YEDDYGF G SNWVFG HYSEGLD F GGTRLT SWGQGV H VL VVTVSS Y S E G E DS115DM2\301045646.1PATENT Docket No. Y7969-99073 RJrl inR MB EVQLVES 76 A 188 DIVMTQ 296 VQ 398 5.053E- NA 000000 120 8_A NA C GGGLVQP R TPLSLPI TIA 10 106 9E- pex GGSLRLS N TPGEPAS FPY 07 GT6 CVASGFT S ISCRSSQ S_wk FSNYDIH L SLLHSN10_0 WVRQAP T GNTYLH20 GKGLEW Y WYLQKPVAVTSYD Y GQSPQL GSKKYY E LIYGGSN ADSVKD D RASGVP RFTISRD D DRFSGS NSKNML Y GSGTDF YLQMNN G TLKISKV LQLEDTA D EAEDVG VYYCAR Y VYYCVQ NSLTYYE Y TIAFPYS DDYGDY T FGQGTK YTFDIVY F VEIK YFDYWG D QGVPVTV I SS V Y Y F D Y RJrl mR MB EVQLVES 77 A 189 QVILTQS 297 QM 399 2.194E- NA NA 8.84 8_A NA C GGGLAKP K PATLSLS HS 09 4E- pex GGSLRLS D PGDRAT YSS 07 GT6 CAASGFT L LSCRAS LT_wk FSSYVMH D QSVSSY10_0 WVRQAP Y LAWYQ21 GKGLEW E QKPGRAVSVISSG D PRLLIYG GTTHYAE D ASSRAT SVKGRFT Y GIPDRFS ISRDNSK G GSGSGT NTFSLQM Y EFTLTIS NNLRAED Y SLEPEDF TAVYYC D AVYFCQ AKDLDY T MHSYSS EDDYGY E LTFGGG YDTEGG G TKVEIK MRYYGL G DSWGQG M VVVTVST R Y Y G L D S RJrl mR MB QVQLVQS 78 A 190 QSVLTQ 298 LA 400 1.532E- NA 6.196E- 7.47 8_A NA C GAEVKKP R PPSVSGA WD 09 06 8E- pex GASVKLS E PGQRVTI NSL 06 GT6 CKASGYI E SCTGSSS SV_wk FTSYSLN L NIGSSYV NIWVRQAP G SWYQQFGQGLEW L PGTVPK116DM2\301045646.1PATENT Docket No. Y7969-99073 10_0 MGWINPS F LLIIIENN22 NGNTGY E KRPSGVAQKFQG Y SDRFSGS RVTMTR Y KSGTSA DTSTSTA E SLTITGL YMELSSL D QPGDEA RSEDTAV D DYYCLA YYCAREE Y WDNSLS LGLFEYY G VNILGA EDDYGYF Y GTRLTV SPSSSGL F L DSWGQG S VVVTVSS PSssG L D S RJrl mR MB EVQLVES 79 A 191 DIVMTQ 299 MQ 366 3.447E- NA NA 4.11 8_A NA C GGGLAKP K TPLSLPV TLQ 09 9E- pex GGSLRLS D TPGEPAS TPL 06 GT6 CAASGFT K ISCRSSQ T_wk FSSYAMH T SLLDSD10_0 WVRQAP P GYTHLH23 GKGLEW V WYLQKPVSGISSG Y GQSPQL GSTIYAD Y LMYLVS SVKGRFT E NRASGV ISRDNSK D PDRFSGS NTLSLQM D GSGTDF NSLRAED Y TLK1SRVTAVYYC G EAEDVG AKDKTPV Y IYYCMQ YYEDDY Y TLQTPLT GYYFTGD F FGGGTR SGGVWD T VEIK YWGQGV G LVTVSS D S G G V W D Y RJrl mR MB QVQLVQS 80 T 192 QSMLTQ 300 SA 401 6.865E- NA NA NA 8_A NA C GTEVKKP R PPSVSAA WD 08pex GASVKLS G PGQRVTI SSLGT6 CKASGYT A SCSGSSS SV_wk FTSYSIN M NIGISYV GL10 0 WVRQAP A SWYQQV24 GQGLEW S PGTVPKMGWINPT P LLIFQDN NGNTGY E KRPSGV AQKFQG Y SDRFSGS RVTMTR Y KSGTSADTSTSTA E SLAITGL117DM2\301045646.1PATENT Docket No. Y7969-99073 YMELSSL D QTGDEA RSEDTAI D DYYCSA YYCTRG Y WDSSLS AMASPEY V VGLFGG YEDDYV Y GTRLTV YFFSGIDE F L FDSWGQ F GVLVTVS S A G I D E F D S RJrl mR MB QVQLVQS 81 A 193 DIQMTQ 301 QH 402 1.419E- NA 2.695E- 8.14 8_A NA C GTEVKKP R SPSSLSA YY 09 06 3E- pex GASVKLS G SVGDRV GTP 08 GT6 CKASGYT G TITCRAS FT_wk FTSYSIS V QGITND10_0 WVRQAP S LAWYQ25 GQGLEW S QKPGETMGWINPS E PKLLIYE NGNTVY D ASSLQS AQKFQG D GIPSRFS RVTMTR Y GSGSGT DTSTSTA D DFTLTIS YMDLSSL Y SLQSEDF RSEDTAV Y ATYYCQ YYCARG F HYYGTP GVSSEDD T FTFGPGT YDYYFTE E RLDIK ELITE YG E LDSWGQ L GVVVTVS T S T F Y G L D S RJrl mR MB EVQLVES 82 A 194 QSAPTQ 302 CSF 403 6.254E- NA 3.552E- 6.28 8_A NA C GGGLVQP R PPSVSGS TTS 11 06 E- pex GGSLRLS D PGQSVTI STF 09 GT6 CAASGFT S SCTGTSS L_wk FRSYSMH L DIGYYN10_0 WVRQAP K AVSWYQ26 GKGLEW F QQPGTAVAIISFDG Y PKLMIY SKNYYA E GVSNRP DSVKDRF D SGVSDR TISRDNS D FSGSKSG KNMLYL Y NTASLTI QMNNLK D SGLQAE LEDTAVY Y DEADYY YCARDSL Y CCSFTTS KFYEDDY S STFLFGGDYYSFQE F118DM2\301045646.1PATENT Docket No. Y7969-99073 DTGGGPD Q GTRLTV YWGQGV E L LVTVSS D T G G G P D Y RJrl mR MB QVQLVQS 83 A 195 DIVMTQ 303 MQ 363 2.153E- NA 5.429E- 6.89 8_A NA C GAEVKKP R TPLSLPV TLQ 11 06 3E- pex GASVKLS F TLGEPAS TPF 09 GT6 CKASGYP G ISCRSSQ T_wk FTSYSIT R SLLSSDG10_0 WVRQTP R YNYLN27 GQGLEL Y WYLQKPMGWINP Y GQSPHL RDGDTG E LIYYGSN YAQKFQ D RASGVP GRVTLTR D DRFSGS DESTSTA Y GSDTDF YMEVSSL D TLKISRV RSDDTAV Y EAEDVG YYCARFG Y IYYCMQ RRYYED Y TLQTPFT DYDYYYI I FGPGTK SGTTDHY S LDIK GLDSWG G QGVVVT T VSS T D H Y G L D S RJrl mR MB QVQLQES 84 A 196 EVVFTQ 304 QS 404 5.746E- NA NA 6.44 8_A NA C GPGLVKP R PIISVSGS YD 09 E- pex SETLSLT G PGQTVTI SY 07 GT6 CAVSGGS R SCTRSSG NLF_wk ISGYWW P SIDSEYV10_0 GWIRQPP R QWYQQ28 GKGLEWI G RPGSAPTGYIGGSS A TVIYEH GSTNYSP N NQRPSG SLKSRVTI Y VPDRFS SIDTSKN E GSIDSSS QVSLKLN D NSASLAI SVTAADT D SGLKSE AVYFCAR Y DEADYY GRPRGAN G CQSYDS YEDDYG Y YNLFFG YWTFITG W GGTRLT SDWYFD T VL LWGPGEP F HISSIT119DM2\301045646.1PATENT Docket No. Y7969-99073 G S D W Y F D L RWj mR MB EVQLAES 85 A 197 DIVMTQ 305 MQ 405 3.841E- NA NA 1.75 18_ NA C GGGLVQP R TPLSLPV NIQ 11 SE Ape GGSLRLS H TPGEPAS LPY 05 xGT CAASGFT R ISCRSSQ S6_w FSSYEMQ G SLLDSEDkl0_ WVRQAP R GSTYLD029 GKWLES II WYLQKPVSVISGD L GQSPQL SSNTHYA Y LIYDVSN DSVKGRF E RASGVP TISRDNA D DRFSGS KNSLSLQ D GSDADF MNSLRAE Y ILKISRV DTAVYY A EAEDVG CARHRG Y VYYCM RHLYEDD Y QNIQLPY YAYYYS Y SFGQGT GGSLDV S KVEIK WGRGVL G VTVSS G S L D V RWj mR MB VEQLVES 86 L 198 DIVMTQ 306 MQ 406 5.691E- NA NA 2.38 18_ NA C GGGLVQP R TPLSLPV GIE 10 8E- Ape GASLRLS G TPGEPAS FPY 08 xGT CAASEFT S ISCRSSQ S6_w FSSYDMH V SLLDSEGkl0_ WVRQAP G GNTYLD030 GKGLEW G WYLQKPVSAISIAG E GQSPQP DTRYPDS D LIYEVSN VKGRFTI D RASGVP SRDNAK Y DRFSGS NSLYLQ G GSDTDF MNSLRAE Y ILKISRV DTAIYYC F EAEDVG LRGSVGG Y VYYCM EDDYGYF M QGIEFPY YMSPTNR S SFGQGT FDVWGP P KVEIK GVLVTVS L S N R F DV120DM2\301045646.1PATENT Docket No. Y7969-99073 RWj inR MB QVQLQES 87 A 199 QSAPTQ 307 YS 407 1.558E- NA NA 544 18_ NA C GPGVVKP R PPSVSGS YTS 09 7E- Ape SETLSLT D PGQSVTI SST 07 xGT CAVSGGS L SCTGTSS WV6_w ISSGYYS S DVGGYNklO_ WSWIRQP L YVSWYQ031 PGKGLE Y QHPGKAWMGRIH H PKLMIY GDGENT E GVSNRP NYSPSLK D SGVSDR SRITISKD D FSGSKSD TSKNQFS Y NTASLTI LKLSSVT D SGLQAE AADTAV Y DEADYY YYCARD Y CYSYTSS LSLYHED Y STWVFG DYDYYY T GGTRLT TDIYIRD D VL YWGQGV I LVTVSS Y I R D Y RWj mR MB VEQLVES 88 A 200 QSALTQ 308 GS 408 7.491E- NA 000001 416 18_ NA C GGGLVQP R PPSVSKS YRS 11 935 5E- Ape GASLRLS G LGQSVTI GET 06 xGT CAASEFT Q SCTGTSS YI6_w FSTYDMH A DIGGYNkl0_ WVRQAP Y GVSWYQ032 GKGLEW Y QHSGTALSGITIGG E PRLLIYE DTYYPDS D VNKRPS VRGRFTI D GVSDRF SRDNGK Y SGSKSG NSLYLQ D NTASLTI MNSLRAE Y SGLQAE DTAVYY I DEADYY CARGQA P CGSYRS YYEDDY T GTTYIFG DYIPTEQ E AGTRLT SKYALDS Q VL WGQGVV sVTVSS K Y A L D S RWj mR MB EVQLVES 89 A 201 DIVMTQ 309 MQ 409 6.696E- NA 5431E- 376 18_ NA C GGGLVQP K TPLSLPV SAE 10 06 2E- Ape GGSLRLS D TPGEPAS FPL 07 xGT CAASGFT R ISCRSSQ T6_w FSSYGMY V SLLDSDkl0_ WVRQAP V GYTRLD033 GKGLEWI F WYLQKPSAINSGG Y GQSPQL GNTYYA E E1YEVSNDSVKGRF D RASGVP121DM2\301045646.1PATENT Docket No. Y7969-99073 TISRDNS D DRFSGS KNTLSLQ Y GSGSYF MNSLRVE D TLKISRV DTAVYY Y EAEDVG CAKDRV Y VYYCM VFYEDDY S QSAEFPL DYYSLVS L TFGGGT PYNRFDV V KVDIK WGAGVL S VTVSS P Y N R F D V RWi mR MB QVQLQES 90 A 202 DIVMTQ 310 MQ 410 4.832E- NA NA 779 18_ NA C GPGLVKP R TPLSLPV HK 10 4E- Ape SETLSLT Y TPGEPAS ALP 07 xGT CAVSGGS P ISCRSSQ LT6_w ISSNYWT S SLLHTDklO_ WIRQSPG P GYTYLD034 KGLEWIG P WYLQKPYVYGGS D GQSPQL GFTNYNP Y LIYGGST SLESRVTI E RASGVP STDTSKN D DRFSGS QFSLKLS D GSGTDF SLTAADT Y TLKISKV AIYYCAR N EAEDVG YPSPPDY Y VYYCM EDDYNY Y QHKALP YSTPPGP S LTFGGG PLDSWG T TKVEIK QGVVVT P VSS P G P P L D S RWj mR MB QVQLQES 91 A 203 QSAPTQ 311 CSY 411 2.76E-09 NA NA 2.28 18_ NA C GPGLVKP R PPSVSGS TAS E- Ape SETLSLT D PGQSVTI STV 07 xGT CTVSGGS R SCTGTSS F6_w ISNYYWS P DIGYNNkl0_ WIRQPPG V VVSWYQ035 KGLEWIG L HHPGTANIDANSA Q PKLMIY GTNYNPS Y GVSYRP LKSRVTIS S SGVSDR KDTSNNQ D FSGSKSG FSLKLNS Y NTASLTI VTAADT E SGLQAE ALYYCA D DEADYY RDRPVLQ D CCSYTA YSDYEDD Y SSTVFFGYTYYYN T122DM2\301045646.1PATENT Docket No. Y7969-99073 GVYFDY Y GGTRLT WGQGVL Y VL VTVSS Y N G V Y F D Y RWj niR MB EVQLVES 92 A 204 QPVLTQ 312 QT 412 4.072E- NA 4018E- 227 18_ NA C GGGLAKP K SPSASAS WT 08 06 3E- Ape GGSLRLS V LGASVK TGI 07 xGT CAASGFT S LTCTLSS QV6_w FSSYWMS Q GIISSYNIklO_ WVRQAP E AWHQQ036 GKGLEW D QQGKAPVSDINTG Y RFLMWL GDSTFYA E KIDGSHS DSVKGRF D KGDGIP TISRDNS D DRFSGSS KNTLSLQ Y SGAERY MNSLRAE G LTISNLQ DTAVYY H SEDEAD CAKVSQE Y YYCQTW DYEDDY Y TTGIQVF GHYYTFE T GGGTRL SWGYGV F TVL DSWGQG E VVVTVSS S W G Y G V D S RWj mR MB QVQLQFS 93 A 205 QAALTQ 313 SSY 413 7435E- NA NA NA 18_ NA C GPGLVKP S PRSVSGS AG 10Ape SETLSLT A PGQSVTI SNTxGT CAVSGGS I SCAGTSS LV6_w FSSSWW E DIGGYNkl0_ GWIRQPP G YVSWYQ037 GKGLEW S QHPGTAVOTINGS G PKLMIY GGSNYLN S EVNKRP PSLKSRV E SGVSDR TLSVDTS Y FSGSKSG KNQLSLR Y NTASLTI LSSVTAA E SGLQAE DTAVYFC E DEADYY ASAIEGS D CSSYAG GSEYYEE Y SNTLVF DYGYYQ G GGGTRL TFGVGYF Y TVL DYWGQG Y VL VTVSS QTF123DM2\301045646.1PATENT Docket No. Y7969-99073 G V G Y F D Y RWj mR MB QVQLQES 94 A 206 DVVMTQ 314 MQ 414 2.381E- NA 000001 1 91 18_ NA C GPGLVKP R SPLSLPIT GT 10 686 9E- Ape SETLSLT H PSQPASI HLP 08 xGT CAVSGDS H SCRSSQS YS6_w FSGYFWG G LVHSDGklO_ WVRQPP N KTYLSW038 GKGLEWI W YQQKPGGFISGGS A QPPRRLL GSTDYNP G YQVSTR SLKSRVTI S DSGVPD STDTSKS S RFSGGG QFSLRLT D AGTDFT SVTAADT Y LKISRVE AVYYCA Y AEDVGV RHHGNW E YYCMQ AGSSDYY D GTHLPY EDDYGYF D SFGQGT YTGDSLD Y KVEIK VWGRGIL G VTVSS Y F Y T G D S L D V RWj niR MB EVQLVES 95 V 207 QAGLTQ 315 SA 415 5.134E- NA 7508E- 553 18_ NA C GGGLVQP K PPSVSKG WE) 10 06 7E- Ape GGSLRLS D LRQTAT RSL 08 xGT CAASGFT G LTCTGN SA6_w FSSYGMY R SNNVGN WVkl0_ WVRQAP T QGAAW039 GKGLEWI Y LQQHQGSAINSGG Y HPPKLLS DSTYYAD E YRNNNR SVKGRFT D PSGISER ISRDNSK D FSASRSG NTLALQ Y DTASLTI MNSLRVE G TGLQPE DTAVYY D DEADYY CVKDGR Y CSAWDR TYYEDD Y SLSAWV YGDYYIE I FGGGTR GVLPTAD E LTVL NGLDSW G GQGVVV V TVSS LPT124DM2\301045646.1PATENT Docket No. Y7969-99073 A D N G L D S RWj mR MB QVTLKES 96 A 208 QPVLTQ 316 QT 416 1.457E- NA NA 1 02 18_ NA C GPALVKP R SPSASAS GET 10 4E- Ape TQTLTLT V LGASVK GM 08 xGT CTFSGFSI A LTCTLSS L6_w TTAGTGV V GHSNYNklO_ GWIRQPP I. IAWHQQ040 GKALEW Q QQGKAPLAAIYWII Y RYLMW DSKYYST L LKSDGS SLKSRLTI D HSKGDG YTDTSRN S IPDRFSG QVVLTM T SSSGAER TNMDPV A YLTISNL DTATYYC Y QSEDEA ARVAVL E DYYCQT QYLDSTA D GTTGML YEDDYG D FGGGTR YSYTVSP Y LTVL YFDYWG G QGVLVT Y VSS S Y T V S P Y F D Y RWj mR MB EVQLVES 97 G 209 DVVMTQ 317 GQ 417 2219E- NA 4.966F- 1 00 18_ NA C GGGLVQP R SPLSLPIT GA 09 06 9E- Ape GGSLTLS E PGQPASI HLP 06 xGT CAASGFT A SCRSSQS FT6_w FSSYGMY T LLHSNGkl0_ WVRQAP P NTYLNW041 GKGLEWI M YQQKPGSGINNSG Y QPPRLLI DSTYYAD Y YQVSNR SVKGRFT E YSGVPD ISRDNSK D RFSGSG NTLSLQM D AGTDFT NSLRAED Y LKISRVE TAMYYC G AEDIGIY GREATPM Y YCGQGA YYEDDY Y HLPFTFG GYYSLE S PGTKLDI WGDSGG L K QVDYFDF E WGQGVL W VTVSS GD125DM2\301045646.1PATENT Docket No. Y7969-99073 S G G Q V D Y F D F RWj niR MB QVQLQES 98 A 210 DIVMTQ 318 MQ 369 3.979E- NA NA 286 18_ NA C GPGLVKP G TPLSLPV GIQ 10 6E- Ape SETLSLT A TPGEPAS LPY 08 xGT CTVSGAS G ISCRSSQ S6_w ISSNWWS G SLLDSEDklO_ WIRQPPG P GNTYLD042 KGLEWIA G WYLQKPKINGYIG E GQSPQL DTDYNPS G LIHEVSN LKSRVTIS Y RASGVP KDASKN E DRFSGS YFSLKLG D GSDTDF SVTAADT D TLKISRV AVYYCA Y EAEDVG GAGGPGE G VYYCM GYEDDY Y QGIQLPY GYHYTFE H SFGQGT TKHRKD Y KVEIK NRFDVW T GVGVLV F TVSS E T K H R K D N R F D V REt Prot MB EVHLVES 99 A 211 DIVMTQ 319 MQ 418 6.384E- NA 1 144E- 553 18_ ein C GGGLVQP K TPLSLPV GLE 11 06 2E- Ape GGSLRLS A TLGEPAS FP 08 xGT CAASGFT S ISCRSSQ WT6_lo FSTYGMY G SLLNIEDngH WVRQAP F GNTYLE3_W GKGLEWI S WYLQKPK10 SGINTGG S GQSPQR001 GSTYYAD G LIYDVSNSVKGRFT W RASGVP ISRDNSK Y DRFSGS NTLSLQM T GSDTDF NSLRVED Y TLKISRV TAVYYC E EAEDVG AKASGFS D VYYCM SGWYTY D QGLEFPEDDYGPE Y126DM2\301045646.1PATENT Docket No. Y7969-99073 YYYGLDS G WTFGQG WGQGVV P TKVEIK VTVSS E Y Y Y G L D S REt Prot MB QVTLKES 100 A 212 SSGLTQE 320 GS 419 1.306E- NA NA 283 18_ ein C GPPLVKP R AALSVA WD 09 4E- Ape TQTLTLT V LGHTVR NS 06 xGT CTFSGFSI P MTCQGD GN6_lo STTGTGV A SLKTYY QYngH GWIRQPP T ASWYQQ V3_W GKALEW F KPGQVPK10 LASIYWN L VLVIYG_002 DNKYYN G NTNRPSTSLESRL D GIPGRFS TISTDAS F GSWSGN KNQVFLT Y TGSLTIT MTNLDP E GAQVED VDTATY D EADYYC YCARVPA D GSWDNS TFLGDFY Y GNQYVF EDDYVY V GAGTRL YYTDCRF Y TV DFWGQG Y VL VTVSS Y T D C R F D F REt Prot MB EVQLVES 101 A 213 QSVETQ 321 QS 420 1 961 E- NA NA NA 18_ ein C GGGLAKP K PPSVSGA YD 10Ape GGSLTLS D PGQRVTI SDLxGT CAASGFT G SCTGSSS LA6_lo FSSYWM V NIGGYY VLngH NWVRQIP Y VQWYQ3_W GKGLEWI D QLPRTAK10 STMDSGG Y PKLLIYE_004 GRTYYA E NHKRPSGSVKGRF D GVSDRF TVSRDNS D SGSQSG KNTLSLQ Y TSASLTI MNSLRAE D TGLQSE DTAVYY L DEADYY CAKDGV L CQSYDS YDYEDD L DLLAVL YDLLLPL P FGGGTR TTTNYFD L LTV YWGQGV T LVTVSS TTN127DM2\301045646.1PATENT Docket No. Y7969-99073 Y F D Y REt Prot MB EVQLVES 102 A 214 DLVMTQ 322 MQ 421 1.447E- NA 3022E- 284 18_ ein C GGGLVQP K 1PLSLPV 1LQ 11 07 8E- Ape GGSLRLS G TPGEAA IPW 10 xGT CAASGFT S SISCRSS T6_lo FSTYDMF R QSLLDSngH WVRQAP P DGYTHL3_W GKGLEWI Y HWYLQK10 SAINSGG Y KPGQSP_005 GSTYYVD F. QLLISLGSVQGRFT D SNRASG ISRDNSK D VPDRISG NTLSLQM Y SGSGTD NSLRPED G FTLKISR TAVYYC D VEAEDV AKGSRPY Y GVYYC YEDDYG Y MQTLQI DYYFQGE F PWTFGQ YGLDSW Q GIKVEI GQGVVV G K TVSS E Y G L D SRiol Prot MB QVTLKES 103 A 215 QAALTQ 323 NS 422 5.144E- NA 1.655E- 324 8_A ein C GPALVKP R PPSVSGS YA 10 06 3E- pex TQTLTLT G PGQSVTI GS 07 GT6 CTFSGFS P SCTGSSS NTLIon LTISGMG R DIGGYN LgH3 VGWIRQP E YVSWYQ_W PGKALE E QYPGKAK10 WLALIY Y PKLMIY_007 WDDDF. R Y DVTKRPYSTSLKN E SGVSDR RLTISKD D FSGSKSG TSKNQVV E NTASLTI LTMTNM Y SGLQAE DPMDTA G DEADYY TYYCAR Y CNSYAG GPREEYY D SNTLLFG EDEYGY Y GGTRLT DYTAQW T VL GAFDFW A GQGLRVT Q VSS wG A F DF128DM2\301045646.1PATENT Docket No. Y7969-99073 Riol Prot MB QVQLMQ 104 A 216 QAALTQ 324 SSY 423 9.268E- NA NA 108 8_A ein C SGAEVKK R PRSVSGS EGS 09 3E- pex PGASVKL G PGQSVTI NSL 06 GT6 SCKASGY L SCTGTSS VIon TFNIYAII Q DIGGYNgH3 WVRQAP G YVSWYQ_W GQGLEW E QHPGTAK10 MGGIIPL V PKLMIY_008 VGITSYA G DVSERPSQKFQGR E GVSDRF VHTADT D SGSKSG STSTAYM D NTASLTI ELSSLRSE Y SGLQAE DTAVYFC G DEADYY ARGLQGE Y CSSYEGS VGEDDY Y NSLVFG GYYYTSG Y GGTRLT YGLDSW T VL GQGVVV S TVSS G Y G L D SRiol Prot MB EVQLAES 105 A 217 DIVMTQ 325 MQ 424 1.454E- NA NA 365 8_A ein C GGGLVQP R TPLSLPV SVE 10 1E- pex GGSLRLS G TPGEPAS YP 09 GT6 CAASGFT R ISCRSSQ YTIon FTGYEM S SLFDGDgH3 HWVRQA Y SANTYL_W PGKGLES Y DWYLQK10 VSVIGGD E KPGQSP_009 NSYTRYA D QLLIYMDSVKGRF D LSNRAS TISRDNA Y GVPDRF KNSLSLQ G TGSGSG MNSLRA F TDFTLKI ADTAVY Y SRVEAE YCARGRS S DVGVYY YYEDDY D CMQSVE GFYSDRD R YPYTFG YYGLHS D LGTKVEI WGQGVV Y K VTVSS Y G L H S RPz Prot MB QVTLKES 106 A 218 QSVLTQ 326 QS 425 1.692E- NA 1442E- 222 18_ ein C GPALVKP R PPSVSGA YD 09 06 1E- Ape TQTLTLT V PGQRVT NRL 06 xGT CTFSGFSI L MSCTGS SV6_lo STTGTGV P SSNIGGY HIngH GWIRQPP L YVQWY3_W GKALEW G QQLPGTK10 LASIFWN E APKLLIF016 DNKYYN1 D ENNKRPSPKSRLTI Y SGASDR129DM2\301045646.1PATENT Docket No. Y7969-99073 FTDTSKN E FSGSQSG QVVLTM D TSASLTI TNMDPA D TGLQSE DTATYYC Y DEADYY ARVLPLG E CQSYDN EDYEDD Y RLSVHIF YEYIYTE I GTGTRL RGAAFDF Y TVL WGQGLR T VTVSS E R G A A F D F RPz Prot MB QVTLKES 107 A 219 QSVLTQ 327 QS 426 7.379E- NA 1 291E- 703 18_ ein C GPALVKP R PPSVSGA YD 11 06 9E- Ape TQTLTLT V PGQRVTI SNL 08 xGT CTFSGFSI V SCTGSSS SV6_lo TETGEGV P NIGGYY HVngH GWIRQPP L VQWYQ3_W GKALEW G QLPGTAK10 LASIFWN E PKLLIYE_017 DNKYYNI G NNKRPSSPKSRLTI Y GVSDRF FTDSSKN E SGSQSG QVVLTM D TSASLTI TNMDPA D TGLQSE DTGTYYC Y DEADYY ARVVPLG E CQSYDS EGYEDD Y NLSVHV YEYFYTE F FGAGTR RGAAFDF Y LTVL WGQGLR T VTVSS E R G A A F D F RPz Prot MB QVQLVQS 108 A 220 EIVLTQP 328 QS 427 3.802E- NA 000003 163 18_ ein C GAEVKKP R HSVSGSP AD 10 052 5E- Ape GASVKLS M GQTVTIS DS 07 xGT CKASGYT G CSGSIDS YN6_lo FTSYSIN L EYVQW VLngH WVRQAP E YQQRPG3_W GQGLEW V NAPTTVIK10 MGWINPS Y YKDNQR018 NGNIGYA E PSGVPDQKFQGR D RFSGSID VTMTRD D SSSNSAS TSTSTAY Y LAISGLK MELNSLR G SEDEAD SEDTAVY Y YYCQSAYCARMG Y DDSYNV130DM2\301045646.1PATENT Docket No. Y7969-99073 LEVYEDD T LFGGGT YGYYTGI G RLTVL KGLFVDY I WGQGVL K VTVSS G L F V D Y RPz Prot MB EVQLVES 109 A 221 DIVMTQ 329 MQ 428 6.51E-11 NA NA 7.50 18_ ein C GGGLVQP K TPLSLPV GIE 7E- Ape GGSLRLS G TPGEPAS FP 08 xGT CAASGFT V ISCRSSQ WT6_lo FSSYGMY R SLLDSEDngH WVRQAP Y GSTYLE3_W GKGLEWI Y WYLQKPK10 SGIDSGG E GQSPQP019 GNTYHA D LIYEVSNDSVKGRF D RASGVP TISRDNS Y DRFSGS KNTLSLQ D GSDTDF MNSLRVE T TLKISRV DTAVYY Y EAEDVG CAKGVR Y VYYCM YYEDDY T QGIEFP DTYYTRG R WTFGQG VNSLDV G TKVEIK WGRGVL V VTVSS N S L D V REt Prot MB QVTLKES 110 A 222 DIVMTQ 330 MQ 429 5.51E-11 NA 2.895E- 3 13 18_ ein C GPALVKP R TPLSLPV TLQ 08 1E- Ape TQTLTLT V TPGEPAS TP 06 xGT CTLSGFSI N ISCRSSQ WT6_lo TAIGAGV L SLLDSDngH GWIRQPP P GYTIILII3_W GKALEW G WYLQKPK17 LATIYWN V GQSPQL_023 DSKYSST S LVYLGSSLKSRLSI P NRASGV STDASKN P PDRFSGS QVVLTM D GSGTAF TNMDPV N TLKISRV DTATYFC D EAEDVG ARVNLPG E VYYCM VSPPDND Y QTLQTP EYDYVYF D WTFGQG YTAGFLD Y TKVEIK HWGQGV V LVTVSS Y F Y TAG131DM2\301045646.1PATENT Docket No. Y7969-99073 F L D H REt Prot MB QVTLKES 111 A 223 SSGLTQE 331 AS 430 1.445E- NA NA 2 17 18_ ein C GPALVKP R PALSVA WD 09 4E- Ape TQTLTLT V LGHTVR NS 08 xGT CTFSGFSI T MTCQGD GN6_lo TTTGTGV A SLKTYY HFHngH GWIRQPP T ASWYQQ3_W GKALEW C KPGQVPK17 LANIYWN V VLVIYG_024 DSKYYNT G NTNRPSSLKSRLTI D GIPGRFS STDTSKN Y GSWSGN LVILTMT Y TGSLTIT NMDPVD E GAQVED TATYHCA D EADYYC RVTATCV E ASWDNS GDYYED Y GNHHIF EYVCYDT V GTGTRL ECRFDN C TVL WGQGAL Y VTVSS D T E C R F D N REt Prot MB QVTLKES 112 A 224 QSALTQ 332 GS 431 1.37E-10 0.00 000002 650 18_ ein C GPALAKP R PPSVSKS YRS 0016 363 9E- Ape TQTLTLT A LGQSVTI GY 2 09 xGT CTFSGFSI G SCTGTSS TFI6_lo STTGTGV I DIGGYNngH GWIRQPP A AVSWYQ3_W GKALEW G QDSGTAK17 LASIYWH T PRLLIYE_026 DSKYYNT N VSKRPSSLKNRLT G GVSDRF IFTDASK D SGSKSG NQVVLT Y NTASLTI MTNMDP E SGLQAE VDTATY D DEADYY YCARAGI D CGSYRS AGTNGD Y GYTFIFG YEDDYD D FGTRLTI YSAFFSA Y L FDFWGQ S GRRVTVS A S F F S A F DF132DM2\301045646.1PATENT Docket No. Y7969-99073 REt Prot MB QVQLVQS 113 A 225 DIQMTQ 333 QII 432 1.102E- NA NA 000 18_ ein C GAEVKKP R SPSSLSA GY 09 001 Ape GASVKLS L SVGDRV GTP 094 xGT CEASGYA E TITCRAS LT6_lo FSIYGISW G ENVNNYngH VRQAPG A LNWYH3_W QGLEWM F QKPGKAK17 GGIIPRVG D PKLLIYK_028 ITTYAQK Y ASTLQSFQGRVTI E GVPSRFS TADTSTS D GSGSGT TAYMELS D DYTFTIS SLRSEDT Y SLQPED AVYYCA G VATYYC RLEGAFD L QHGYGT YEDDYG L PLTFGQ LLFESKS F GTKVEI NSLDVW E K GQGVLV S TVSS K S N S L D V RPz Prot MB EVQLVES 114 V 226 EIVMTQ 334 VQ 433 1.327E- NA 3336E- 107 18_ ein C GGGVVQ R TPLSLPI TIA 10 06 4E- Ape PGGSLRL G TPGEPAS FPF 06 xGT SCAASGF G ISCRSSQ T6 lo TFDDYA G SLLHSDngH MHWVRQ G GDTRLH3_W APGKGLE L WYLQKPK17 WVSGISW V GQSPQL_033 SGDGTNY H LIYGGSSADSVKG E RVSGVP RLTISRD D DRFSGS NAKNSLY D GSGTDF LQMNSLR Y TLKISKV VEDTAV E EAEDVG YYCVRG Y VYYCVQ GGGLVH Y TIAFPFT EDDYEY Y FGPGTK YYTDANP T LDMK LDSWGQ D GVVVTVS A S N P L D S RPz Prot MB EVQLVES 115 V 227 EIVMTQ 335 MQ 434 7.859E- NA 1211E- 703 18_ ein C GGGLVQP K TPLSLPV AIQ 12 07 6E- Ape GGSLRLS D TPGEPAS LPF 11 xGT CVASGFT P ISCRSSQ S6_lo FSSYGIY R NLLASEngH WVRQAP E DGNTYL3_W GKGLEWI Y DWYLQSIISSGGD H KPGQSP133DM2\301045646.1PATENT Docket No. Y7969-99073 K17 KTIIYADS E QLLIYEV_034 VKGRFTI D SNRASGSRDNSKN D VPDRFS TVSLQIN Y GSGSDT SLRAEDT E DFTLKIS AVYYCV Y RVEAED KDPREYH Y VGVYYC EDDYEY H MQAIQL YHLTGG L PFSFGQG DRFDVW T TKVEIK GAGVLV G TVSS G D R F D V RPz Prot MB EVQLLES 116 A 228 EMVMT 336 QQI 435 3.458E- NA NA 1 14 18_ ein C GGGVVQ R QSPATLS SN 11 SEApe PGGSLRL D LSPGER WP OS xGT SCAASGF R ATLSCR HPT6_lo TFDDYA S TSLSVSNngH MHWVRQ P SLAWYQ3_W APGKGLE K KKPGQAK17 WVSVIA V PRLLIYG_035 WSGDNT H ASSRATVYADSV E GIPDRFS KGRFTIS D GSGSGT RDNAKN D EFTLIISR SLFLQMN Y LEPEDV RLRTEDT D GVYYCQ GFYYCAR Y QISNWP DRSPKVH Y HPTFGG EDDYDY S GTKVEI YSTEDYG T K LDVWGQ E GVVVTVS D S Y G L D V REt Prot GC QVTLKES 117 A 229 ETVVTQ 337 ML 356 3.345e-06 NA 2628E- NA 18_ ein GPALVKP R EPSLSVS YM 06Ape TQTLTLT A PGGTVT GSxGT CTFSGFSI T LTCGLSS GIL6_lo STTGTGV G GSVSTS LngH GWIRQPP E NYPSWY3_W GKALEW E QQTPGQK4_ LASIYWN Q APRTLIY007_ DSKYYST G STNTRPSiGL SLKSRLTI Y GVPDRFSTDTSKN Y SGSILGN QVVLTM E KAALTIT TNMDPV D GAQADD DTATYYC D ESDYYC ARATGEE Y MLYMGS QGYYED G GILLFGGDYGYYY Y134DM2\301045646.1PATENT Docket No. Y7969-99073 TEEDVRF Y GTRLTV DVWGAG Y L VLVTVSS T E E D V R F D V RPb Prot GC EVQLVES 118 A 230 DIVMTQ 338 MQ 436 7.901e-09 NA 000002 143 18_ ein GGGLAKP K TPLSLPV SIF. 061 4e- Ape GGSLRLS G TPGEPAS FPF 06 xGT CAASGFT W ISCRSSQ T6_lo FSSYAMH G SLLDSDngH WVRQAP G GYTCLD3_W GKGLEW A WYLQKPK4_ VSAISSG G GQSPQL011_ GSTYYAD E LIYEVSNiGL SVKGRFT D RVSGVPISRDNSK D DRFSGS NTLSLQM Y GSGTDF NSLRAED G TLKISRV TAVYYC Y EAEDVG AKGWGG Y VYYCM AGEDDY S QSIEFPF GYYSFLG F TFGPGT DYNSLDV L KLDIK WGRGVL G VTVSS D Y N S L D V RF.t Prot GC EVQLVES 119 A 231 QPVLTQ 339 QT 437 NA NA NA NA 18_ ein GGGLVQP K SPSASAS WTApe GGSLRLS D LGASVK TGIxGT CAASGFT E LTCTLSS RV6_lo FSSYGMY L GHSSYAIngH WVRQAP E AWHQQ3_W GKGLEWI L QQGKAPK8_ SAINSGG Y RYLMRL012_ GSTYYAD I NSVGSHiGL SVKGRFT P SKGDGIPISRDNSK K DRFSGSS NTLSLQM V SGAERY NSLRAED S LTISNLQ TAVYYC N SEDEAD AKDELEL E YYCQTW YIPKVSN D TTGIRVF EDDYGYS D GGGTRL KILEGDY Y TVL WGQGVL G VTVSS YSK135DM2\301045646.1PATENT Docket No. Y7969-99073 I L E G D Y REt Prot GC EVQLVES 120 A 232 QPVLTQ 339 QT 437 NA NA NA NA 18_ ein GGGLVQP K SPSASAS WTApe GGSLRLS D LGASVK TGIxGT CAASGFT Q LTCTLSS RV6_lo FSSYGMY L GHSSYAIngH WVRQAP E AWHQQ3_W GKGLF. WI L QQGKAPK8_ SAINSGG Y RYLMRL013_ GSTYYAD I NSVGSIIiGL SVKGRFT P SKGDGIPISRDNSK K DRFSGSS NTLSLQM V SGAERY NSLRAED S LTISNLQ TAVYYC N SEDEAD AKDQLEL E YYCQTW YIPKVSN D TTGIRVF EDDYGY D GGGTRL YYTLDG Y TVL DYWGQG G VLVTVSS Y Y Y T L D G D YRiol Prot GC QVQLVQS 121 A 233 DIQMTQ 340 QH 438 1.832e-08 NA NA 6.23 8_A ein GAEVKKP R SPSSLSA SY 9e- pex GASVKLS G SVGDRV GTP 06 GT6 CKASGYT P TITCRAS LTIon FSIYAISW D ENVNNYgII3 VRQAPG V LIIWYQ_W QGLEWM V QKPGKAK8_ GGIIPLVG A PKLLIYA014_ ITNYAQK D ASTLQSiGL FQGRVTI Y GVPSRFSTADTSTS E GSGSGT TAYMELS D DFTLTIS SLRSEDT D SLQPED AVYYCA Y VATYYC RGPDVV G QHSYGT ADYEDD Y PLTFGG YGYYYR Y GTKVEI PEDNYFD Y K IWGPGTPI R TISS P E D NYF136DM2\301045646.1PATENT Docket No. Y7969-99073 D IRiol Prot GC QVQLVQS 122 A 234 DIQMTQ 340 QH 438 1.996e-07 NA NA NA 8_A ein GAEVKKP R SPSSLSA SYpex GASVKLS G SVGDRV GTPGT6 CKASGYT P TITCRAS LTIon FSIYAISW D ENVNNYgH3 VRQAPG V LHWYQ_W QGLEWM V QKPGKAK8_ GGIIPLVG A PKLLIYA015_ ITNYAQK D ASTLQSiGL FQGRVTI Y GVPSRFSTADTSTS E GSGSGT TAYMELS D DFTLTIS SLRSEDT D SLQPED AVYYCA Y VATYYC RGPDVV G QHSYGT ADYEDD Y PLTFGG YGYYYR Y GTKVEI PENWYF Y K DIWGPGT R PITISS P E N W Y F D IRiol Prot GC QVQLVQS 123 A 235 QAALTQ 341 SSK 439 4.568e-06 NA NA NA 8_A ein GAEVKKP R PPSVSGS TGSpex GASVKLS G PGQSVTI NTLGT6 CKASGYT G SCTGTSS VIon FSIYAISW G DIGGYNgH3 VRQAPG T YVSWYQ_W QGLEWM Y QHPGKAK8_ GGIIPLVG E PKLMIY016_ ITNYAQK D DVSKRPiGL FQGRVTI D SGVSDRTADTSTS Y FSGSKSG TAYMELS G NTASLTI SLRSEDT Y SGLQAE AVYYCA Y DEADYY RGGGTYE Y CSSYAG DDYGYY T SNTLVF YTDGQY D GGGTRL YYDRTY G TVL HFDYWG Q QGVLVT Y VSS Y Y D R T YHF137DM2\301045646.1PATENT Docket No. Y7969-99073 D YRiol Prot GC QVQLVQS 124 A 236 QAALTQ 342 SSY 423 1.986e-06 NA NA NA 8_A ein GAEVKKP R PRSVSGS EGSpex GASVKLS G PGQSVTI NSLGT6 CKASGYT L SCTGTSS VIon FSIYAISW G DIGGYNgH3 VRQAPG P YVSWYQ_W QGLEWM S QHPGTAK8_ GGIIPLVG I PKLMIY018_ ITNYAQK E EVSKRPSiGL FQGRVTI T GVSDRFTADTSTS E SGSKSG TAYMELS D NTASLTI SLRSEDT D SGLQAE AVYYCA Y DEADYY RGLGPSIE G CSSYAG TEDDYG Y SNTLVF YYYTSEY Y GGGTRL NSLDVW Y TVL GRGVLVT T VSS S E Y N S L D VRiol Prot GC EVQLVES 125 A 237 DIVMTQ 343 MQ 363 1.541e-08 NA 4437E- 105 8_A ein GGGLVQP R TPLSLPV TLQ 06 9e- pex GGSLRLS M TPGEPAS TPF 07 GT6 CAASGFT G ISCRSSQ TIon FSSYGMH R SLLDSDgH3 WVRQAP R GYTHLH_W GKGLEW Y WYLQKPK8_ VAVISYD Y GQSPQL024_ GSKKYY E LIYLVSNiGL ADSVKD D RASGVPRFTISRD D DRFSGS NSKNML Y GSGTDF YLQMNN G TLKINRV LKLEDTA Y EAEDVG VYYCAR Y VYYCM MGRRYY Y QTLQTPF EDDYGY F TFGPGT YYFAGDP A KLDIK PLLDVW G GRGVLVT D VSS P P L L DV138DM2\301045646.1PATENT Docket No. Y7969-99073 Riol Prot GC EVQLVES 126 A 238 QAALTQ 258 SSY 364 7.435e-06 NA NA 1.14 8_A ein GGGLVQP R PRSVSGS AG Oe- pex GGSLRLS M PGQSVTI SNT 05 GT6 CAASGFT G SCTGTSS FYIIon FSSYGMH R DIGGYNgH3 WVRQAP R YVSWYQ_W GKGLEW Y QHPGTAK8_ VAVISYD Y PKLMIY025_ GSKKYY E EVSKRPSiGL ADSVKD D GVSDRFRFTISRD D SGSKSG NSKNML Y NTASLTI YLQMNN G SGLQAE LKLEDTA Y DEADYY VYYCAR Y CSSYAG MGRRYY Y SNTFYIF EDDYGY F GAGTRL YYFAGDS A TVL PLLDVW G GRGVLVT D VSS S P L L D VRiol Prot GC EVQLVES 127 A 239 DIVMTQ 344 MQ 441 2.771e-09 NA 000000 624 8_A ein GGGLVQP R TPLSLPV SIE 56 7e- pex GGSLRLS D TPGEPAS FP 07 GT6 CAASGFT T ISCRSSQ WTIon FSSYGMH R SLLDSDgH3 WVRQAP Y GYTCLD_W GKGLEW Y WYLQKPK8_ VAVISYD E GQSPQL026_ GSKKYY D LIYEVSNiGL ADSVKD D RVSGVPRFTISRD Y DRFSGS NSKNML G GSGTDF YLQMNN Y TLKISRV LKLEDTA Y EAEDVG VYYCAR Y VYYCM DTRYYED I QSIEFPW DYGYYYI F TFGQGT FAGRIYG A KVE1KLDSWGQ G GVVVTVS R S I Y G L D SRiol Prot GC EVQLVQS 128 A 240 DIVMTQ 345 MQ 366 4.880e-07 NA NA NA 8_A ein GAEVKRP K TPLSLPV TLQpex GESLKISC D TPGEPAS TPLGT6 KTSGYSF V ISCRSSQ TIon TSYWISW N SLLDSDgH3 VRQMPG G GYTHLHW KGLEWM D WYLQKP139DM2\301045646.1PATENT Docket No. Y7969-99073 K8_ GAIDPSD Y GQSPQL028_ SDTRYSP E LIYLVSNiGL SFQGQVT D RASGVPISADKSIS D DRFSGS TAYLQW Y GSGTDF SSLKASD G TLKINRV SATYYCA Y EAEDVG KDVNGD Y VYYCM YEDDYG Y QTLQTP YYYTVG T LTFGGG RGHYFD V TKVEIK YWGQGV G LVTVSS R G H Y F D Y RGp Prot GC EVQLAES 129 A 241 DIVMTQ 346 MQ 367 6.187e-08 NA 000003 268 18_ ein GGGLVQP R TPLSLPV SVE 284 6e- Ape GGSLRLS G TPGEPAS YPF 06 xGT CAASGFT R ISCRSSQ T6_lo FSGYEMH M SLFDSDngH WVRQAP Y YANTYL3_W GKGLESV Y DWYLQK12 SVIGGDS E KPGQSP_035 SYTHYAD D QLLIYMJG SVKGRFT D LSNQAS L ISRDNAK Y GVPDRF NSLSLQM G SGSGSG NSLRAAD Y TDFTLKI TAVYYC Y SRVEAE ARGRMY Y DVGVYY YEDDYG T CMQSVE YYYTGD G YPFTFGP YYGLDS D GTKLDI WGQGVV Y K VTVSS Y G L D S RGp Prot GC EVQLAES 129 A 241 DIVMTQ 346 MQ 367 6.187e-08 NA 000003 268 18_ ein GGGLVQP R TPLSLPV SVE 284 6e- Ape GGSLRLS G TPGEPAS YPF 06 xGT CAASGFT R ISCRSSQ T6_lo FSGYEMH M SLFDSDngH WVRQAP Y YANTYL3_W GKGLESV Y DWYLQK12 SVIGGDS E KPGQSP_036 SYTHYAD D QLLIYMJG SVKGRFT D LSNQAS L ISRDNAK Y GVPDRF(036 NSLSLQM G SGSGSGiGL NSLRAAD Y TDFTLKIis TAVYYC Y SRVEAEthe ARGRMY Y DVGVYYsam YEDDYG T CMQSVE140DM2\301045646.1PATENT Docket No. Y7969-99073 e as YYYTGD G YPFTFGP035_ YYGLDS D GTKLDIiGL) WGQGVV Y KVTVSS Y G L D S RJzl Prot GC QCVEQLV 131 A 242 DIVMTQ 347 MQ 442 2.765e-09 NA 3.042E- 06 8_A ein ESGGGLV R TPLSLPV GIQ 06 7e- pex QPGASLR G TPGEPAS FPY 07 GT6 LSCAASE G ISCRSSQ SIon FTFSSYD R SLLDSFDgH3 MHWVRQ G GNTYLD_W APGKGLE G WYLQKPK12 WVSAISI Y GQSPQL_038 GGGTYYP E LIYEVSNJG DSVKGRF D RASGVP L TISRDNA D DRFSGS KNSLYLQ Y GSDTDF MNSLRAE G TLKISRV DTAVYY Y EAEDVG CARGGR Y VYYCM GGYEDD Y QGIEYPY YGYYYFI F SFGQGT GGRRSLD I KVEIK VWGRGV G LVTVSS G R R S L D V RJzl Prot GC QCVEQLV 132 A 243 DIVMTQ 347 MQ 442 2.995e-08 NA 9.676E- 06 8_A ein ESGGGLV R TPLSLPV GIQ 06 2e- pex QPGASLR G TPGEPAS FPY 06 GT6 LSCAASE G ISCRSSQ SIon FTFSSYD R SLLDSEDgH3 MHWVRQ G GNTYLD_W APGKGLE G WYLQKPK12 WVSAISI Y GQSPQL_039 GGGTYYP E LIYEVSNJG DSVKGRF D RASGVP L TISRDNA D DRFSGS KNSLYLQ Y GSDTDF MNSLRAE G TLKISRV DTAVYY Y EAEDVG CARGGR Y VYYCM GGYEDD Y QGIEYPY YGYYYIV I SFGQGT GGRRSLD V KVEIK VWGRGV G LVTVSS G R RSL141DM2\301045646.1PATENT Docket No. Y7969-99073 D V RJzl Prot GC EVQLVES 133 A 244 DIQMTQ 348 1.464e-06 NA NA 9.10 8_A ein GGGLVQP R SPSSLSA 6e- pex GGSLRLS D SVGDRV 06 GT6 CAASGFT G TITCRASIon FSSYGMH T QGISNYLgH3 WVRQAP A SWYQQK_W GKGLEW Y PGKAPKK12 VAVISYD Y RLIYAAS_040 GSKKYY E SLESGVPJG ADSVKD D SRFSGSGI. RFTISRD D SGTEFTLNSKNML Y TISSLQP YLQMNN G EDFAAY LKLEDTA Y YCLQYN VYYCAR Y SKPFTFG DGTAYY S PGTKLDI EDDYGY F K YSFNECN N SLDVWG E RGVLVTV C SS N S L D V RJzl Prot GC EVQLVES 134 A 245 DIQMTQ 348 3.333e-07 NA 1.326E- 06 8_A ein GGGLVQP R SPSSLSA 06 6e- pex GGSLRLS D SVGDRV 07 GT6 CAASGFT G TITCRASIon FSSYGMH T QGISNYLgH3 WVRQAP A SWYQQK_W GKGLEW Y PGKAPKK12 VAVISYD Y RLIYAAS_041 GSKKYY E SLESGVPJG ADSVKD D SRFSGSG L RFTISRD D SGTEFTL NSKNML Y TISSLQP YLQMNN G EDFAAY LKLEDTA Y YCLQYN VYYCAR Y SKPFTFG DGTAYY S PGTKLDI EDDYGY F K YSFNDEY N NSLDVW D GRGVLVT E VSS Y N S L D V RJzl Prot GC EVQLVES 135 A 246 DIQMTQ 348 8.948e-07 NA NA NA 8_A ein GGGLVQP R SPSSLSApex GGSLRLS D SVGDRVGT6 CAASGFT G TITCRASIon FSSYGMH T QGISNYL142DM2\301045646.1PATENT Docket No. Y7969-99073 gII3 WVRQAP A SWYQQK_W GKGLEW Y PGKAPKK12 VAVISYD Y RLIYAAS_042 GSKKYY E SLESGVP_iG ADSVKD D SRFSGSGL RFTISRD D SGTEFTL NSKNML Y TISSLQP YLQMNN G EDFAAY LKLEDTA Y YCLQYN VYYCAR Y SKPFTFG DGTAYY S PGTKLDI EDDYGY F K YSFNDYN N SLDVWG D RGVLVTV Y SS N S L D V REt Prot GC EVQLVES 136 A 247 DIVMTQ 349 MQ 372 3.588e-08 NA 1.779e- NA 18_ ein GGGLVQP K TPLSLPV ALE 05 Ape GGSLRLS D TLGEPAS FPYxGT CAASGFT R ISCRSSQ S6_lo FSSYGMY S SLLDSEDngH WVRQAP P GNTYLE3_W GKGLEWI Y WYLQKPK12 SAINSGG E GQSPQL_033 GSTYYAD D LIYEVSN_iG SVKGRFT D RASGVPL ISRDNSK Y DRFSGS NTLSLQM G GSDTDF NSLRAED Y TLKISRV TAVYYC Y EAEDVG AKDRSPY Y VYYCM EDDYGY T QALEFP YYTGLDR G YSFGQG FDVWGA L TKVEIK GVLVTVS D S R F D V REt Prot GC EVQLVES 136 A 247 DIVMTQ 349 MQ 443 3.588e-08 NA 1 779e- NA 18_ ein GGGLVQP K TPLSLPV GLE 05 Ape GGSLRLS D TLGEPAS FPYxGT CAASGFT R ISCRSSQ T6_lo FSSYGMY S SLLDSEDngH WVRQAP P GNTYLE3_W GKGLEWI Y WYLQKPK12 SAINSGG E GQSPQL_034 GSTYYAD D LIYEVSNJG SVKGRFT D RASGVP L ISRDNSK Y DRFSGS(034 NTLSLQM G GSDTDFiGL NSLRAED Y TLKISRVis TAVYYC Y EAEDVGthe AKDRSPY Y VYYCMsam EDDYGY T QALEFP143DM2\301045646.1PATENT Docket No. Y7969-99073 e as YYTGLDR G YSFGQG0.33 FDVWGA L TKVEIKiG GVLVTVS DL) S RF D V REt Prot GC EVQLVES 138 A 162 DIVMTQ 350 MQ 444 7.046e-07 NA 4.205e- NA 18_ ein GGGLVQP N TPLSLPV ALE 06 Ape GGSLRLS L TLGEPAS FPxGT CAASGFT A ISCRSSQ WT6_lo FSSYGMY T SLLDSEDngH WVRQAP Y GNTYLE3_W GKGLEWI E WYLQKPK4_ SAINSGG D GQSPQL008_ GSTYYAD D LIYEVSNiGL SVKGRFT Y RASGVPISRDNSK G DRFSGS NTLSLQM Y GSDTDF NSLRAED Y TLKISRV TAVYYC Y EAEDVG ANLATYE T VYYCM DDYGYY S QALEFP YTSINRF I WTFGQG DVWGAG N TKVEIK VLVTVSS R F D V REt Prot GC EVQLVES 139 A 248 DIVMTQ 351 MQ 445 4.250e-06 NA 4.334e- NA 18_ ein GGGLVQP K TPLSLPV GIE 06 Ape GGSLRLS D TPGEPAS YPLxGT CAASGFT T ISCRSSQ T6 lo FSSYGMY T SLLDSEDngH WVRQAP Y GNTYLE3_W GKGLEWI Y WYLQKPK4_ SAINSGG E GQSPQP009_ GSTYYAD D LIYEVSNiGL SVKGRFT D RASGVPISRDNSK Y DRFSGS NTLSLQM G GSDTDF NSLRAED Y TLKISRV TAVYYC Y EAEDVG AKDTTY R VYYCM YEDDYG R QGIEYPL YYRRTY T TFGGGT NSLDVW Y KVEIK GRGVLVT N VSS S L D V REt Prot GC EVQLVES 139 A 248 DIVMTQ 351 MQ 446 4.250e-06 NA 4.334e- NA 18_ ein GGGLVQP K TPLSLPV GIQ 06 Ape GGSLRLS D TPGEPAS FPLxGT CAASGFT T ISCRSSQ T6_lo FSSYGMY T SLLDSEDngH WVRQAP Y GNTYLE144DM2\301045646.1PATENT Docket No. Y7969-99073 3_W GKGLEWI Y WYLQKPK4_ SAINSGG E GQSPQP010_ GSTYYAD D LIYEVSNiGL SVKGRFT D RASGVP(010 ISRDNSK Y DRFSGSiGL NTLSLQM G GSDTDFis NSLRAED Y TLKISRVthe TAVYYC Y EAEDVGsam AKDTTY R VYYCMe as YEDDYG R QGIEYPL009_ YYRRTY T TFGGGTiGL) NSLDVW Y KVEIKGRGVLVT N VSS S L D VRiol Prot GC QCVEQLV 140 A 249 DIVMTQ 352 MQ 376 1.871e-06 NA 9.120e- NA 8_A ein ESGGGLV R TPLSLPV GIQ 07 pex QPGASLR G TPGEPAS LPLGT6 LSCAASE S ISCRSSQ TIon FTFSSYD E SLLHSGgH3 MHWVRQ A GKTYLY_W APGKGLE Y WYLQKPK8_ WVSGISI E GQSPQL021_ GGGTYYP D LIYEVSNiGL DSVKGRF D RASGVPHSRDNA Y DRFSGS KNSLYLQ G GSGTDF MNSLRAE Y TLKISRV DTAVYY Y EAEDVG CARGSEA Y VYYCM YEDDYG T QGIQLPLYYYTRHS R TFGGGT LDVWGR H KVEIK GVLVTVS S S L D VRiol Prot GC QCVEQLV 140 A 249 DIVMTQ 352 MQ 440 1.871e-06 NA 9.120e- NA 8_A ein ESGGGLV R TPLSLPV SIQ 07 pex QPGASLR G TPGEPAS LPLGT6 LSCAASE S ISCRSSQ TIon FTFSSYD E SLLHSGgH3 MHWVRQ A GKTYLY_W APGKGLE Y WYLQKPK8_ WVSGISI E GQSPQL022_ GGGTYYP D LIYEVSNiGL DSVKGRF D RASGVP(022 HSRDNA Y DRFSGSiGL KNSLYLQ G GSGTDFis MNSLRAE Y TLKISRVthe DTAVYY Y EAEDVGsam CARGSEA Y VYYCMe as YEDDYG T QGIQLPL021_ YYYTRHS R TFGGGTiGL) LDVWGR H KVEIKSL145DM2\301045646.1PATENT Docket No. Y7969-99073 GVLVTVS D S V RJzl Prot GC EVQLVES 141 A 250 DIVMTQ 353 MQ 440 1.152e-05 NA NA NA 8_A ein GGGLVQP K TPLSLPV SIQpex GGSLRLS D TPGEPAS LPLGT6 CAASGFT R ISCRSSQ TIon FSSYGMY Q SLLDSEDgH3 WVRQAP E GNTYLD_W GKGLEWI Y WYLQKPK12 SAINSGG Y GQSPQL_043 GSTYYAD E LIYEVSNJG SVKGRFT D RASGVPI. ISRDNSK D DRFSGSNTLSLQM Y GSDTDF NSLRAED G TLKISRV TAVYYC Y EAEDVG AKDRQE Y VYYCM YYEDDY Y QGIEYPL GYYYTG T TFGGGT ASLIWGQ G KVEIK GVLVTVS A S S L I RJzl Prot GC EVQLVES 141 A 250 DIVMTQ 354 2.486e-08 NA NA NA 8_A ein GGGLVQP K TPLSLPVpex GGSLRLS D TPGEPASGT6 CAASGFT R ISCRSSQIon FSSYGMY Q SLLDSEDgH3 WVRQAP E GNTYLD_wk GKGLEWI Y WYLQKP12_0 SAINSGG Y GQSPQL44_i GSTYYAD E LIYEVSNGL SVKGRFT D RASGVP ISRDNSK D DRFSGS NTLSLQM Y GSDTDF NSLRAED G TLKISRV TAVYYC Y EAEDVG AKDRQE Y VYYCM YYEDDY Y QGIQLPL GYYYTG T TFGGGT ASLIWGQ G KVEIK GVLVTVS A S S L I RJzl Prot GC EVQLVES 141 A 250 DIVMTQ 355 5.147e-06 NA 1.169e- NA 8_A ein GGGLVQP K TPLSLPV 05 pex GGSLRLS D TPGEPASGT6 CAASGFT R ISCRSSQIon FSSYGMY Q SLLDSEDgH3 WVRQAP E GNTYLD_wk GKGLEWI Y WYLQKP12_0 SAINSGG Y GQSPQL45_i GSTYYAD E LIYEVSNGL SVKGRFT D RASGVP ISRDNSK D DRFSGSNTLSLQM Y GSDTDF146DM2\301045646.1PATENT Docket No. Y7969-99073 NSLRAED G TLKISRV TAVYYC Y EAEDVG AKDRQE Y VYYCM YYEDDY Y QALEFPL GYYYTG T TFGGGT ASLIWGQ G KVEIK GVLVTVS A S S L I RJzl Prot GC QCVEQLV 142 A 251 DIVMTQ 353 MQ 440 7.158e-06 NA 1.702e- NA 8_A ein ESGGGLV R TPLSLPV SIQ 05 pex QPGASER G TPGEPAS LPEGT6 LSCAASE R ISCRSSQ TIon FTFSSYD R SLLDSEDgH3 MHWVRQ Y GNTYLD_W APGKGLE Y WYLQKPK8_ WVSAISI E GQSPQL030_ GGGTYYP D LIYEVSNiGL DSVKGRF D RASGVPTISRDNA Y DRFSGS KNSLYLQ G GSDTDF MNSLRAE Y TLKISRV DTAVYY Y EAEDVG CARGRR Y VYYCM YYEDDY T QGIEYPL GYYYTE E TFGGGT DYGLDS D KVEIK WGQGVV Y VTVSS G L DS

[0459] The invention is further described by the following numbered paragraphs:1. A non-naturally occurring engineered immunogen for inducing or eliciting broadly neutralizing antibodies against HIV, comprising a modified recombinant HIV envelope (Env) protein comprising a mutation in the V1 / V2 region, said mutation comprising K121T, T128K, and / or K168R relative to the BG5O5 MD39 sequence, and wherein said modified Env protein induces B cells expressing antibodies that bind to the apex epitope of HIV Env..2. The immunogen of paragraph 1, wherein the modified Env protein further comprises glycosylation sites at positions N241 and N289. The immunogen of paragraph 1 or 2, wherein the modified Env protein further comprises one or more mutations that promote proper protein folding and / or trimer stability.147DM2\301045646.1PATENT Docket No. Y7969-99073 3. The immunogen of paragraph 1 or 2, wherein the modified Env protein further comprises one or more mutations that facilitate expression or native-like conformation and / or enhance trimer stability.4. The immunogen of any one of paragraphs 1-3, wherein the mutation comprises K121T, T128K, and / or K168R, wherein the positions are numbered according to the sequence of BG505 MD39, and wherein the sequence of BG505 MD39 is: AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGE LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQ AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVAIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 4).5. The immunogen of any one of paragraphs 1-4, wherein the modified Env protein comprises glycosylation sites at positions N241 and / or N289, wherein the positions are numbered according to BG505 MD39, and wherein the sequence of BG505 MD39 is: AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGE LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQ AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVAIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH148DM2\301045646.1PATENT Docket No. Y7969-99073 LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 4).6. The immunogen of any one of paragraphs 1-5, wherein the modified Env protein is ApexGT6 comprising ApexGT6 congly or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the sequence of ApexGT6 congly, wherein the sequence of ApexGT6 congly is: AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVSTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVGLQCTNVTNNITDDMRG ELKNCSFNATTELRNKRQKVYSLFYRLDIVPMVDLWTNYRLISCNTSAITQACPKVSFEP IPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIR SENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSK ATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNST WISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRD GGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAV AIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHW GIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWL QWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 5).7. The immunogen of any one of paragraphs 1-6, wherein the modified Env protein is a soluble trimer.8. The immunogen of any one of paragraphs 1-6, wherein the modified Env protein is a membrane-bound trimer.9. The immunogen of paragraph 8, wherein the modified Env protein is ApexGT6 L14 gpl51 having the sequence:AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVSTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVGLQCTNVTNNITDDMRG ELKNCSFNATTELRNKRQKVYSLFYRLDIVPMVDLWTNYRLISCNTSAITQACPKVSFEP IPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRS ENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKA149DM2\301045646.1PATENT Docket No. Y7969-99073 TWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTW ISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDG GSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSG SGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWL WYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 6).10. A nucleic acid molecule encoding the immunogen of any one of paragraphs 1-9.11. The nucleic acid molecule of paragraph 10, wherein the nucleic acid is an mRNA.12. A composition comprising the immunogen of any one of paragraphs 1-9 or the nucleic acid molecule of paragraph 10 or 11, and a pharmaceutically acceptable carrier.13. The composition of paragraph 12, wherein the nucleic acid molecule is an mRNA formulated in a lipid nanoparticle (LNP).14. A method of inducing an immune response against HIV, comprising administering to a subject an effective amount of the immunogen of any one of paragraphs 1-9 or the composition of paragraph 12 or 115. The method of paragraph 14, wherein the immunogen is administered as an adjuvanted protein.16. The method of paragraph 14, wherein the immunogen is administered as an mRNA-LNP.17. The method of any one of paragraphs 14-16, further comprising administering one or more booster immunizations.18. The method of any one of paragraphs 14-17, wherein the immune response comprises induction of B cells expressing antibodies having heavy chain complementarity determining region 3 (HCDR3) lengths of 24 amino acids or greater.150DM2\301045646.1PATENT Docket No. Y7969-99073 19. The method of any one of paragraphs 14-18, wherein the immune response comprises induction of B cells expressing antibodies that utilize the IGHD3-15 gene segment.20. The method of any one of paragraphs 14-19, wherein the immune response comprises the induction of B cells expressing antibodies containing a DDY motif.21. A method of producing antibodies that bind to the apex epitope of HIV Env, comprising: (a) administering an engineered immunogen to a subject to induce production of B cells expressing antibodies that bind to the apex epitope of HIV Env, wherein the immunogen comprises a modified HIV Env protein comprising, consisting essentially of or consisting of mutation(s) that enhance binding to precursors of broadly neutralizing antibodies (bnAbs) targeting the apex epitope of HIV Env; (b) isolating the induced B cells from the subject after administering the immunogen; and (c) producing monoclonal antibodies from the isolated B cells22. The method of paragraph 21, wherein the engineered immunogen comprises, consists essentially of, or consists of an immunogen of any one of paragraphs 1-9.23. An isolated antibody or antigen-binding fragment thereof that binds to the apex epitope of HIV Env, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR3 of 24 amino acids or greater in length.24. The isolated antibody or antigen-binding fragment thereof of paragraph 23 induced or elicited by an immunogen of any one of paragraphs 1-9.25. The isolated antibody or antigen-binding fragment of paragraph 23 or 24, wherein the heavy chain utilizes the IGHD3-15 gene segment.26. The isolated antibody or antigen-binding fragment of any one of paragraphs 23-25, wherein the antibody or antigen-binding fragment binds to ApexGT6 with a KD of 10 nM or less.27. A pharmaceutical composition comprising the immunogen of any one of paragraphs 1-9 and a pharmaceutically acceptable carrier, or comprising the composition of paragraph 12 or 13, or an antibody elicited or induced by the immunogen of any one of paragraphs 1-9, or an antibody of any one of paragraphs 23-26, or an antibody obtained by a method of paragraph 21 or 22.151DM2\301045646.1PATENT Docket No. Y7969-99073 28. A kit comprising the immunogen of any one of paragraphs 1-9, or the composition of paragraph 12 or 13, or an antibody elicited or induced by the immunogen of any one of paragraphs 1-9, or an antibody of any one of paragraphs 23-26, or an antibody obtained by a method of paragraph 21 or 22, and instructions for administration.29. A kit comprising the immunogen of any one of paragraphs 1-9, or the composition of paragraph 12 or 13, or an antibody elicited or induced by the immunogen of any one of paragraphs 1-9, or an antibody of any one of paragraphs 23-26, or an antibody obtained by a method of paragraph 21 or 22, and instructions for use.30. A method of identifying subjects with precursors to apex bnAbs, comprising: (a) obtaining a biological sample from a subject; (b) contacting the biological sample with an engineered immunogen comprising a modified HIV Env protein, wherein the Env protein includes mutations that enhance binding to bnAb precursors specific for the apex epitope of HIV; and (c) detecting binding of B cells in the sample to the immunogen.31. The method of paragraph 30 wherein wherein the engineered immunogen comprises, consists essentially of, or consists of an immunogen of any one of paragraphs 1-9.32. A method of treating or preventing a viral infection in a subject, comprising administering a therapeutically effective amount of the antibody of any one of paragraphs 23-26.33. The method of paragraph 32 wherein the viral infection is an HIV infection.34. A method of detecting HIV infection in a subj ect, comprising: (a) obtaining a biological sample from the subject; (b) contacting the sample with the antibody of any one of paragraphs 23-26; and (c) detecting binding between HIV Env and the antibody.35. A method of identifying antigens capable of eliciting broadly neutralizing antibodies against HIV, comprising: (a) contacting a test antigen with the antibody of any one of paragraphs 23-26; (b) detecting binding between the test antigen and the antibody; and (c) selecting antigens that bind to the antibody as candidates for eliciting broadly neutralizing antibodies152DM2\301045646.1PATENT Docket No. Y7969-99073 36. A method of producing antibodies that bind to the apex epitope of HIV Env, comprising: (a) administering to a subject a nucleic acid molecule encoding the immunogen of any one of paragraphs 1-9; (b) allowing expression of the immunogen in the subject; (c) isolating B cells expressing antibodies that bind to the apex epitope; and (d) producing monoclonal antibodies from the isolated B cells.37. A method of immunizing a subject against HIV infection comprising: (a) administering a first immunization comprising the immunogen of any one of paragraphs 1-9 in a first formulation; and (b) administering one or more boost immunizations comprising the immunogen in a second formulation different from the first formulation38. A method of producing an immune response comprising: (a) administering the mRNA of paragraph 11 formulated in lipid nanoparticles to a subject; (b) allowing expression of the immunogen encoded by the mRNA; and (c) inducing B cells expressing antibodies specific for the apex epitope39. A method of identifying apex epitope-specific B cells comprising: (a) obtaining a biological sample containing B cells; (b) contacting the sample with: (i) a first labeled immunogen comprising the modified Env protein, and (ii) a second labeled immunogen comprising a knockout variant lacking the apex epitope; (c) isolating B cells that bind to the first immunogen but not the second immunogen using flow cytometry sorting.40. A kit for identifying apex epitope-specific B cells comprising: (a) the immunogen of any one of paragraphs 1-9 conjugated to a first detectable label; (b) a knockout variant of the immunogen lacking the apex epitope conjugated to a second detectable label; (c) reagents for cell isolation and flow cytometry; and (d) instructions for identifying B cells that bind specifically to the apex epitope.41. An isolated antibody or antigen-binding fragment thereof that binds to the apex epitope of HIV Env, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR3 sequence according to Table 5.153DM2\301045646.1PATENT Docket No. Y7969-99073 42. The isolated antibody or antigen-binding fragment of paragraph 41, wherein the antibody further comprises a light chain CDR3 sequence according to Table 5.43. The isolated antibody or antigen-binding fragment of paragraph 41 or 42, wherein the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence according to Table 5.References1. P. Anklesaria, N. D. Russell, Iterative hypothesis testing in HIV vaccine research: moving towards success. J Int AIDS Soc 26, e26102 (2023).2. B. F. Haynes, K. Wiehe, S. M. Alam, D. Weissman, K. O. Saunders, Progress with induction of HIV broadly neutralizing antibodies in the Duke Consortia for HIV / AIDS Vaccine Development. Curr Opin HIV AIDS 18, 300-308 (2023).3. T. M. Martin, S. T. Robinson, Y. Huang, Discovery medicine - the HVTN's iterative approach to developing an HIV-1 broadly neutralizing vaccine. Curr Opin HIV AIDS 18, 290-299 (2023).4. R. Govindan, K. E. Stephenson, HIV Vaccine Development at a Crossroads: New B and T Cell Approaches. Vaccines (Basel) 12, (2024).5. X. Xiao, W. Chen, Y. Feng, D. S. Dimitrov, Maturation Pathways of Cross-Reactive HIV- 1 Neutralizin...

Claims

PATENT Docket No. Y7969-99073WHAT IS CLAIMED IS:

1. A non-naturally occurring engineered immunogen for inducing or eliciting broadly neutralizing antibodies against HIV, comprising a modified recombinant HIV envelope (Env) protein comprising a mutation in the V1 / V2 region, said mutation comprising K121T, T128K, and / or K168R relative to the BG5O5 MD39 sequence, and wherein said modified Env protein induces B cells expressing antibodies that bind to the apex epitope of HIV Env.

2. The immunogen of claim 1, wherein the modified Env protein further comprises glycosylation sites at positions N241 and N289.

3. The immunogen of claim 1 or 2, wherein the modified Env protein further comprises one or more mutations that promote facilitate expression or native-like conformation and / or enhance trimer stability.

4. The immunogen of any one of claims 1-3, wherein the mutation comprises K121T, T128K, and / or K168R, wherein the positions are numbered according to the sequence of BG505 MD39, and wherein the sequence of BG505 MD39 is: AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGE LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQ AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVAIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 4).

5. The immunogen of any one of claims 1-5, wherein the modified Env protein comprises glycosylation sites at positions N241 and / or N289, wherein the positions are numbered according to BG505 MD39, and wherein the sequence of BG505 MD39 is: AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGE159DM2\301045646.1PATENT Docket No. Y7969-99073 LKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSL AEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQ AHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNT SGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVAIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 4).

6. The immunogen of any one of claims 1-5, wherein the modified Env protein is ApexGT6 comprising ApexGT6 congly or a variant thereof having at least 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% amino aci sequence identity to the sequence of ApexGT6 congly, wherein the sequence of ApexGT6 congly is: AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVSTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVGLQCTNVTNNITDDMRG ELKNCSFNATTELRNKRQKVYSLFYRLDIVPMVDLWTNYRLISCNTSAITQACPKVSFEP IPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIR SENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSK ATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNST WISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRD GGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAV AIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHW GIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWL QWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 5).

7. The immunogen of any one of claims 1-6, wherein the modified Env protein is a soluble trimer.

8. The immunogen of any one of claims 1-6, wherein the modified Env protein is a membrane-bound trimer.

9. The immunogen of claim 8, wherein the modified Env protein is ApexGT6 L14 gpl51 having the sequence:160DM2\301045646.1PATENT Docket No. Y7969-99073 AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVSTDPNPQEIHLE NVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVGLQCTNVTNNITDDMRG ELKNCSFNATTELRNKRQKVYSLFYRLDIVPMVDLWTNYRLISCNTSAITQACPKVSFEP IPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRS ENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKA TWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTW ISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDG GSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSG SGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQH LLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEI WDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWL WYIKIFIMIVGGLIGLRIVFAVLSVIHRVR (SEQ ID NO: 6).

10. A nucleic acid molecule encoding the immunogen of any one of claims 1-9.

11. The nucleic acid molecule of claim 10, wherein the nucleic acid is an mRNA.

12. A composition comprising the immunogen of any one of claims 1-9 and or the nucleic acid molecule of claim 10 or 11, and a pharmaceutically acceptable carrier.

13. The composition of claim 12, wherein the nucleic acid molecule is an mRNA formulated in a lipid nanoparticle (LNP).

14. A method of inducing an immune response against HIV, comprising administering to a subject an effective amount of the immunogen of any one of claims 1-9 or the composition of claim 12 or 13.

15. The method of claim 14, wherein the immunogen is administered as an adjuvanted protein.

16. The method of claim 14, wherein the immunogen is administered as an mRNA-LNP.

17. The method of any one of claims 14-16, further comprising administering one or more booster immunizations.

18. The method of any one of claims 14-17, wherein the immune response comprises induction of B cells expressing antibodies having heavy chain complementarity determining region 3 (HCDR3) lengths of 24 amino acids or greater.161DM2\301045646.1PATENT Docket No. Y7969-99073 19. The method of any one of claims 14-18, wherein the immune response comprises induction of B cells expressing antibodies that utilize the IGHD3-15 gene segment.

20. The method of any one of claims 14-19, wherein the immune response comprises the induction of B cells expressing antibodies containing a DDY motif.

21. A method of producing antibodies that bind to the apex epitope of HIV Env, comprising:(a) administering an engineered immunogen to a subject to induce production of B cells expressing antibodies that bind to the apex epitope of HIV Env, wherein the immunogen comprises a modified HIV Env protein comprising, consisting essentially of or consisting of mutation(s) that enhance binding to precursors of broadly neutralizing antibodies (bnAbs) targeting the apex epitope of HIV Env;(b) isolating the induced B cells from the subject after administering the immunogen; and(c) producing monoclonal antibodies from the isolated B cells.

22. The method of claim 21 wherein the engineered immunogen comprises, consists essentially of, or consists of an immunogen of any one of claims 1-9.

23. An isolated antibody or antigen-binding fragment thereof that binds to the apex epitope of HIV Env, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR3 of 24 amino acids or greater in length.

24. The isolated antibody or antigen-binding fragment thereof of claim 24 induced or elicited by an immunogen of any one of claims 1-9.

25. The isolated antibody or antigen-binding fragment of claim 23 or 24, wherein the heavy chain utilizes the IGHD3-15 gene segment.

26. The isolated antibody or antigen-binding fragment of any one of claims 23-25, wherein the antibody or antigen-binding fragment binds to ApexGT6 with a KD of 10 nM or less.

27. A pharmaceutical composition comprising the immunogen of any one of claims 1-9 and a pharmaceutically acceptable carrier, or comprising the composition of claim 12 or 13, or an antibody elicited or induced by the immunogen of any one of claims 1-9, or an antibody of any one of claims 23-26, or an antibody obtained by a method of claim 21 or 22.

28. A kit comprising the immunogen of any one of claims 1-9, or the composition of claim 12 or 13, or an antibody elicited or induced by the immunogen of any one of claims 1-9, or162DM2\301045646.1PATENT Docket No. Y7969-99073 an antibody of any one of claims 23-26, or an antibody obtained by a method of claim 21 or 22, and instructions for administration.

29. A kit comprising the immunogen of any one of claims 1-9, or the composition of claim 12 or 13, or an antibody elicited or induced by the immunogen of any one of claims 1-9, or an antibody of any one of claims 23-26, or an antibody obtained by a method of claim 21 or 22, and instructions for use.

30. A method of identifying subjects with precursors to apex bnAbs, comprising:(a) obtaining a biological sample from a subject;(b) contacting the biological sample with an engineered immunogen comprising a modified HIV Env protein, wherein the Env protein includes mutations that enhance binding to bnAb precursors specific for the apex epitope of HIV; and(c) detecting binding of B cells in the sample to the immunogen.

31. The method of claim 30 wherein wherein the engineered immunogen comprises, consists essentially of, or consists of an immunogen of any one of claims 1-9.

32. A method of treating or preventing a viral infection in a subject, comprising administering a therapeutically effective amount of the antibody of any one of claims 23-26.

33. The method of claim 32 wherein the viral infection is an HIV infection.

34. A method of detecting HIV infection in a subject, comprising:(a) obtaining a biological sample from the subject;(b) contacting the sample with the antibody of any one of claims 23-26; and (c) detecting binding between HIV Env and the antibody.

35. A method of identifying antigens capable of eliciting broadly neutralizing antibodies against HIV, comprising:(a) contacting a test antigen with the antibody of any one of claims 23-26;(b) detecting binding between the test antigen and the antibody; and (c) selecting antigens that bind to the antibody as candidates for eliciting broadly neutralizing antibodies.

36. A method of producing antibodies that bind to the apex epitope of HIV Env, comprising:(a) administering to a subject a nucleic acid molecule encoding the immunogen of any one of claims 1-9;163DM2\301045646.1PATENT Docket No. Y7969-99073 (b) allowing expression of the immunogen in the subject;(c) isolating B cells expressing antibodies that bind to the apex epitope; and (d) producing monoclonal antibodies from the isolated B cells.

37. A method of immunizing a subject against HIV infection comprising:(a) administering a first immunization comprising the immunogen of any one of claims 1-9 in a first formulation; and(b) administering one or more boost immunizations comprising the immunogen in a second formulation different from the first formulation.

38. A method of producing an immune response comprising:(a) administering the mRNA of claim 11 formulated in lipid nanoparticles to a subject;(b) allowing expression of the immunogen encoded by the mRNA; and(c) inducing B cells expressing antibodies specific for the apex epitope.

39. A method of identifying apex epitope-specific B cells comprising:(a) obtaining a biological sample containing B cells;(b) contacting the sample with:(i) a first labeled immunogen comprising the modified Env protein, and(ii) a second labeled immunogen comprising a knockout variant lacking the apex epitope;(c) isolating B cells that bind to the first immunogen but not the second immunogen using flow cytometry sorting.

40. A kit for identifying apex epitope-specific B cells comprising:(a) the immunogen of any one of claims 1-9 conjugated to a first detectable label; (b) a knockout variant of the immunogen lacking the apex epitope conjugated to a second detectable label;(c) reagents for cell isolation and flow cytometry; and(d) instructions for identifying B cells that bind specifically to the apex epitope.

41. An isolated antibody or antigen-binding fragment thereof that binds to the apex epitope of HIV Env, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR3 sequence according to Table 5.164DM2\301045646.1PATENT Docket No. Y7969-99073 42. The isolated antibody or antigen-binding fragment of claim 41, wherein the antibody further comprises a light chain CDR3 sequence according to Table 5.

43. The isolated antibody or antigen-binding fragment of claim 41 or 42, wherein the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence according to Table 5.165DM2\301045646.1

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