Design of immunogens that preferentially interact with b cell receptors containing complementary determining region loops of specific composition

Abstract

The present invention provides affinity matured recombinant monoclonal antibodies (mAbs) and fragments that bind specifically to an HLA-E- peptide complex, including HLA-E-VL9 complexes, and regulate the cytotoxicity effector cell function of NK. Herein, monoclonal antibodies were recombinantly derived from isolated functional HLA-E-VL9-specific mAbs from the naïve human B cell repertoire. Such antibodies are capable of regulating effector cell cytotoxicity and can preferentially recognize HLA-E-VL9 peptide complexes expressed on the surface of tumor cells. The monoclonal antibodies were subject to one or more rounds of affinity maturation. The invention provides methods for using affinity matured HLA-E-VL9 mAbs to modulate NK cell function as part of immunotherapeutic strategies.

Description

DESIGN OF IMMUNOGENS THAT PREFERENTIALLY INTERACT WITH B CELL RECEPTORS CONTAINING COMPLEMENTARY DETERMINING REGION LOOPS OF SPECIFIC COMPOSITIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 529,554, filed on July 28, 2023, the entire contents of which are incorporated herein by reference.STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under Center for HIV / AIDS Vaccine Immunology-Immunogen Design grant no. AI144371 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 29, 2024, is named DU8258PCT.xml and is 80,346 bytes in size.TECHNICAL FIELD

[0004] The present invention relates, in general, to a composition suitable for use in inducing antibodies, including anti -HIV- 1 antibodies and, in particular, to immunogenic compositions comprising envelope proteins and nucleic acids to induce cross-reactive neutralizing antibodies and increase their breadth of coverage. The invention also relates to methods of inducing such broadly neutralizing anti -HIV- 1 antibodies using such compositions.BACKGROUND OF THE INVENTION

[0005] The development of a safe and effective HIV-1 vaccine is a high priority of the scientific community working on the HIV-1 epidemic. While anti-retroviral treatment (ART) has dramatically prolonged the lives of HIV- 1 infected patients, ART is not routinely available in developing countries.

[0006] Lineage-based vaccine design, a multi-step vaccination approach that facilitates clonal evolution of an antibody lineage with the ultimate aim of eliciting these target broadly neutralizing antibodies (bnAbs), is one such approach. More traditional routes of vaccination often stimulate and promote neutralization with a single immunogen sometimes administered repeatedly, but these have been largely unsuccessful in eliciting the development of target bnAbs. In contrast, lineage-based vaccinations use a “priming” immunogen to activate target naive B-cell receptors (BCRs), followed by subsequent injections with a series of different “boosting” immunogens optimized to engage with intermediate antibodies and facilitate the process of affinity maturation. This strategy can promote successful maturation of target bnAbs.

[0007] Germline targeting immunogens engage a small number of naturally occurring bnAb precursors. For example, Steichen et al. (Science, 2019) engages precursors at frequencies of 1:54,000,000 naive B cells. Escalano et. al., (Cell, 2016) and Saunders et al. (Science 2019) also showed immunogens interacting with a limited number of naive B cells.

[0008] It is also known that Glycan-V3 bnAbs have long CDR H3 loops, typically greater than 20 amino acids in length. CDR H3 contacts are known to be important for virus neutralization and precursor engagement by germline targeting immunogens. Examples of such bnAbs are DH270.6, BG18, and PGT122.

[0009] Lipocalins are ligand-binding transport proteins that have high specificity for small hydrophobic molecules, like steroids, bilins, retinoids and lipids. Many lipocalins can bind iron. Lipocalins generally have an 8-stranded antiparallel beta barrel with a repeated +1 topology enclosing an internal ligand binding site. Lipocalins can have four hypervariable loops and a deep-barrel pocket.SUMMARY OF THE INVENTION

[0010] Disclosed here are methods for producing immunogens, and specific epitopes, to which certain regions of an antibody can bind. In some embodiments, particular complementarity-determining regions (CDRs) of an antibody can bind to specific epitopes of the immunogen. In some embodiments, a CDR H3-loop of an antibody can bind to an immunogen that has a V3-loop, which can contain surrounding glycans. In some embodiments, the V3-loop can be from HIV-1. However, other CDRs can be used in themethods disclosed herein. Other immunogens can be used in the methods disclosed herein. In some embodiments, the immunogens can be bound by antibodies that are B-cell receptors (BCRs) and the immunogens can activate B-cells or B-cell precursors. In some embodiments, the antibodies can be other receptors. In some embodiments, the B-cells activated by the immunogens can produce broadly neutralizing antibodies (bnAbs). Therefore, the immunogens can function as '‘priming’’ immunogens to target naive BCRs.

[0011] In accordance with a first embodiment, the present invention provides a new prepriming germline targeting immunogens (herein called “primocalins’’) that preferentially bind to B cell receptors containing CDR H3-loops. In some embodiments, the CDR H3-loops are related to those present in DH270 lineage Abs. Other antibodies and other lineages of antibodies can be used.

[0012] In accordance with a second embodiment, the present invention provides compositions and methods for activating B cell receptors containing CDR H3 loops such that there is cell activation.

[0013] In accordance with a third embodiment, the present invention provides methods and compositions for increasing the pool of precursors that the immunogens can engage. In some embodiments, the 10.17DT immunogen can be used.

[0014] In accordance with another embodiment, the present invention provides methods for developing immunogens that bind to the CDR H3 loop of certain antibodies, DH270UCA for example, by selecting for CDR H3 loops which have similar amino acid lengths, and by prioritizing contacts of the immunogen to amino acids in the H3 loops encoded by the D gene.

[0015] In accordance with another embodiment, the present invention provides a primocalin comprising a lipocalin-based protein structure that selects for glycan-V3 precursors comprising target CDR H3 loops.

[0016] In accordance with another embodiment, the present invention provides a method for identifying primocalin binding and iterative selection of modified primocalins with increased binding affinity.

[0017] In accordance with another embodiment, the present invention provides a generalized method of modification of lipocalin to bind antibody sites of interest.

[0018] In accordance with another embodiment, the present invention provides a generalized method of modification of lipocalin to bind CDR H3 loops of HIV antibodies of interest.

[0019] In accordance with another embodiment, the present invention provides a general framework to develop immunogens that aim to elicit antibodies containing functional features of interest, such as long CDR H3 loops, and can be applied to develop immunogens that target the elicitation of other antibodies containing long CDR H3 loops against HIV, influenza, coronaviruses and other infectious agents.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 depicts an example schematic of yeast display workflow for the analysis of primocalin libraries. Library DNA was first cloned into pCTCON2 vectors, then transformed into S. cerevisiae yeast. Prior to FACS screening, constructs were induced with galactose, which resulted in the expression of primocalin proteins on the cell surface. During FACS screening, secondary antibodies bound to c-Myc (FITC-A) when primocalins were expressed on the cell surface, and to a-hlgG (PE- A) when DH270UCA3 or related antibodies interacted with the surface primocalin.

[0021] Figures 2A-2B show an example computational design by Rosetta of initial pnmocalin library templates. (2A) Rosetta computational procedure to identify starting sequences to generate experimental libraries. (2B) Computationally generated model of primocalin-DH270UCA3 complex. Engineered binding contacts (red) of the primocalin (blue) contact the CDR H3 loop (pink) of DH270UCA3 (gray).

[0022] Figures 3A-3B, 3C-3E depict example isolation of primocalin immunogens with broad recognition of DH270UCA3 and related antibodies. (3 A) Eleven DH270UCA3 chimeric antibodies with CDR H3 loops replaced by natural CDR H3 loop displayed binding interactions with a pool of first-generation primocalins at the micromolar level. The D gene region is identical across the sequences, but surrounding regions display significant variety (with deviances marked by the substitute amino acid codes). (3B) The initial library was assessed for binding to GSGN28, 713d, 651a, and 650a antibodies (1 pM). (3C) 713d, 651a and 650a positive-binding cells were next labelled with GSGSN28 (100 nM). (3D) 651a / GSGN28 and 713d / GSGN28 positive cells were then labeled with 713d and 651a,respectively (100 nM each). At each step, cells that fell into the sorting gate (red), were selected by FACS, expanded and used as input for subsequent selection rounds; these cells were PE-A positive (y-axis) and FITC-A positive (x-axis). (3E) Sequence comparison at the four randomized library sites between the original library template (WT) and Poly28-PN, the most frequently observed clone following these sorts.

[0023] Figures 4A-4B, 4C-4D show additional example optimization of Poly28-PN- derived primocalins. (4 A) The Poly28 libraries were first screened with 100 nM DH270UCA3. (4B) Binders were further selected with 1 nM DH270UCA3. (4C) High binding constructs from this pool were screened with 0. 1 nM DH270UCA3. (4D) The four most frequently observed constructs derived from the highest-affinity sorts - Poly281-PN (1), Poly282-PN (2), Poly283-PN (3), Poly284-PN(4) - and the corresponding amino acid substitutions at the four sites selected for variation. The amino acids at these positions of the parent Poly28-PN construct are listed as a reference.

[0024] Figures 5A-5D, 5E-5H depict affinity assays of engineered primocalin constructs. (5 A) SDS-PAGE of expressed and purified primocalin constructs (where 1 corresponds to Poly281-PN, 2 to Poly282-PN, 3 to Poly283-PN, and 4 to Poly284-PN). (5B-5D) ELISA binding of Poly281-PN, Poly283-PN, and Y513 (a previously characterized construct) to target antibodies DH270_Ala, DH270UCA3, and 188 (DH270 related antibodies). (5E) Representative binding traces of Poly281-PN. Poly28-PN (parent Poly28). and Y514 interacting with Dh270UCA3 as measured by SPR techniques. (5F) Extended binding response of Poly281-PN to DH270UCA3 via both direct immobilization and a-hlgG Fc capture. (5G-5H) ka, kd, and KD values calculated for each construct via direct immobilization and a-hlgG Fc capture.

[0025] Figure 6 depicts examples of nanoparticles containing primocalins.

[0026] Figure 7 depicts example measurement of primocalin nanoparticles activating B cells.

[0027] Figure 8A, 8B, 8C depict example immunization studies with primocalins. (8B) Primocalin characterization in vivo in the DH270UCA3 rearranging Rvl mouse model. (8C) Primocalin characterization in vivo - Rvl mice.

[0028] Figure 9 depicts example data from immunization studies with primocalins.

[0029] Figure 10 depicts example data from immunization studies with primocalins.

[0030] Figure 11 depicts example data showing primocalins selecting for CDR H3s.Primocalin Poly 28 selected for CDR H3s using the YYD containing mouse DH2-4 reading frame 3.

[0031] Figure 12 depicts example data relating to primocalins recognizing DH270UCA3 CDR H3 loops. Primocalins recognize diverse DH270UCA3 CDR H3 loop variants.Developed a library of DH270UCA3 antibody variants that contain all the single amino acid mutations in the CDR H3 loop (single site saturation library of DH270UCA3 CDR H3 loop).

[0032] Figure 13 depicts example data showing that engineered primocalins bind diverse CDR H3 loops.

[0033] Figure 14 depicts example data showing that engineered primocalins bind diverse CDR H3 loops.

[0034] Figure 15 depicts example data related to primocalins (Poly28Gl, Y513G1) binding to CDR H3 loops. Primi calins select for CDR H3 loops with the same length, D gene and reading frame as DH270 precursors.

[0035] Figure 16 depicts example data related to primocalins binding to CDR H3 loops. Primocalins select for CDR H3 loops with the same length, D gene and reading frame as DH270 precursors.

[0036] Figure 17 depicts second and third generation primocalins. Engineered primocalins bind DH270UCA3 with high affinity.

[0037] Figure 18 illustrates an example crystal structure ofY513 primocalin.

[0038] Figure 19 depicts example data showing that primocalins recognize antibodies with diverse CDR H3 loops. (19A) shows example structural data. (19B) shows example binding data.

[0039] Figure 20 depicts example binding data.

[0040] Figure 21 illustrates an example crystal structure of DH270UCA3 in complex with Poly 310.

[0041] Figure 22 depicts example immunization studies with primocalins.

[0042] Figure 23 depicts example data illustrating that primocalins can help select forCDR H3 loops specific for anti-HIV broadly neutralizing antibodies (bnAbs). The combination of a primocalin priming immunization and 10. 17 boost is superior to 10.17 alone.

[0043] Figure 24 depicts example data for sequences of 10. 17DT (x4) elicited clones. The data show the diversity of the CDR H3 loops sequences generated.

[0044] Figure 25A-D. (25A-C) depict example data for sequences of Poly28 (x4) elicited clones. (25D) depicts example data for sequences of 10. 17DT (x4) elicited clones. The data show the diversity of the CDR H3 loops sequences generated.

[0045] Figure 26 A-E. (25 A-C) depict example data for sequences of Poly28 (x2) + 10. 17DT (x2) elicited closes. (25D) depicts example data for sequences of 10. 17DT (x4) elicited clones. (25E) depicts example data for sequences of Poly28 (x4) elicited clones.DETAILED DESCRIPTION OF THE INVENTION

[0046] The development of a safe, highly efficacious prophylactic HIV-1 vaccine is important for the control and prevention of HIV-1 infection. A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) (Immunol. Rev. 254: 225-244, 2013). BnAbs are protective in rhesus macaques against SHIV challenge, but as yet, are not induced by current vaccines.

[0047] In accordance with an embodiment, the present invention provides a system for identifying and optimization of a priming immunogen designed to engage the inferred unmutated common ancestor (UCA) of an antibody lineage, here the DH270.6 antibody lineage - a hypothetical construct that can resemble naturally-occurring DH270.6 precursors - and related BCRs. DH270.6 is a well-characterized HIV-1 broadly neutralizing antibody (bnAb) that specifically targets a glycosylated site neighboring the third variable loop (V3) of the HIV-1 envelope glycoprotein (Env), otherwise known as a V3-glycan.8V3-glycan antibodies are particularly potent HIV-1 bnAbs. and require relatively low levels of somatic hypermutation (SHM) to achieve reasonable neutralization breadth (-55%). These properties thus render them attractive targets for vaccine development. Like many other HIV-1 bnAbs, DH270.6 antibodies exhibit unusually long complementarity -determining region-3 (CDR H3) loops that are responsible for a majority of the antigen-binding contacts. The length of these loops allows DH270.6 antibodies to reach through a web of glycans and interact with the HIV-1 Env protein surface. Typically, these long CDR H3 loops are present at low frequencies in the B-cell receptor (BCR) population of healthy individuals. In order to prompt the expansion of the DH270.6 antibody lineage then, a priming immunogen should exhibithigh affinity to the CDR H3 loop of the DH270 UCA construct, as well as to related, naturally occurring BCRs with CDR H3 loops capable of developing into that of the target DH270.6 bnAb. In some embodiments, the strategies disclosed herein, for developing immunogens that bind to specific regions (here, a CDR H3 loop) of antibodies (here, a BCR), can be broadly used for many different types of immunogens, including antigens from many different viruses.

[0048] CDR H3 loops are formed via VDJ recombination, a process by which doublestranded breaks and break repair mechanisms bring genetically encoded segments and non- templated, stochastically generated regions, together. Previously, an immunogen, called 10. 17 DT, was developed to engage DH270UCA. Preliminary analyses indicated that the DH270UCA CDR H3 loop contributed over 50% of the antibody binding contacts in the complex with the viral surface protein. However, binding specific to the DH270UCA3 CDR H3 may restrict interactions with other DH270.6 precursors found in the natural human repertoire, many of which contain diverse amino acids in the CDR H3 loops. While not wishing to be bound by any theory, an immunogen that maintains high affinity to DH270UCA3, but that also interacts with regions of the CDR H3 loop that are conserved across multiple DH270.6 bnAb precursors, will induce broader activation of DH270.6 precursors and create a more robust immune response. Specifically, similar-class BCRs have templated D gene regions that are highly conserved in terms of length, sequence composition, and positional orientation, while regions encoded by A-nucleotide addition typically exhibit high degrees of variation.

[0049] The present invention provides the proof of concept that “priming” immunogens can be made, that are able to bind selectively to amino acids encoded by D genes in the CDR H3 loops of bnAb precursors, including HIV bnAb precursors. In some embodiments, the target is DH270.6. Such immunogens can induce potent immune responses and activate a large number of precursors that can then be matured to bnAbs by additional “boosting” vaccinations.

[0050] Using advanced computational models and a yeast display platform, a new class of immunogens, herein called primocalins, that bind the CDR H3 loops of DH270UCA3 constructs was designed. These primocalin immunogens were derived from lipocalinproteins, which exhibit four hypervariable loops and a deep-barrel pocket, an ideal structural property for engaging lengthy (20 amino acids for DH270UCA3) CDR H3 loops.

[0051] Once this was completed, a multi-step strategy' was employed to further optimize these first-generation primocalin immunogens to recognize diverse BCRs from the natural immune repertoire with high affinity, in order to ensure that upon vaccination, a large number of B cells are activated for subsequent development into the DH270.6 bnAb.

[0052] Step 1. in some embodiments of this process, is an approach to engage DH270UCA3 chimeric antibodies with CDR H3 loops replaced by those of naturally- occurring BCRs with identical D-gene regions, in order to select for primocalins with broad recognition of B-cells in the natural human repertoire, called "poly specific” primocalins. In some embodiments, by isolating first-generation primocalins that are able to bind to different variations of related CDR H3 loops, the number of potential naturally-occurring BCRs that these immunogens are able to activate can be increased. Additionally, on a mechanistic level, in enhancing the breadth of BCRs these primocalin immunogens are able to engage, the probability of isolating constructs that specifically engage the D gene region of the CDR H3 loop is increased. To derive such constructs, first-generation primocalins that exhibited binding to multiple DH270UCA3 chimeric antibodies with varied CDR H3 loops were screened. Constructs were isolated, sequenced, and expressed through E. coli recombination, then assessed for binding affinity via SPR analyses; the Poly28-PN primocalin construct was identified and selected from these steps.

[0053] Previous studies have indicated that bnAb precursor frequencies have an impact on germinal center (GC) responses following immunization. For multivalent immunogens, in some embodiments, an affinity’ threshold of <1 M to target BCRs is necessary to effectively activate precursors present in at least a ~1 : 1 ,000,000 frequency in the natural B-cell repertoire. Even higher precursor frequencies and affinities are required for monomeric immunogens, which have lesser avidity effects compared to polymeric molecules. Further, given that B-cell epitopes are often more complex - rendering them less frequently observed - than T-cell epitopes, and that specific epitopes of naive CD4+ and CD8+ T cells can be present at frequencies ranging from 1 : 1,000,000 - 1 : 100,000, it is reasonable that B-cell frequencies occur at or below the lower limit (1: 1 ,000,000) of this range. While the precise frequencies of DH270.6 precursors and related BCRs in the natural human repertoire areunknown, it is expected that DH270.6 precursor BCRs are rare in the natural human repertoire, prompting for even more enhanced affinity thresholds for effective elicitation of the target BCRs, especially if with monomeric primocalin immunogens. In some embodiments, due to a product of the aforementioned factors, it w as deemed that highly effective primocalin immunogens would display KD values on the nanomolar scale. Initial affinity measurements of the Poly28-PN and DH270UCA3 interaction revealed a ' of -13,000 nM.

[0054] Step 2 of this process, in some embodiments, required that the affinity of the Poly28-PN first-generation construct to DH270.6 related antibodies could be increased in order to robustly activate by vaccination a large number of BCRs from the natural antibody repertoire that resemble DH270UCA3. Through computational protein modeling, libraries of secondary -generation immunogens were created by determining additional sites important for interaction in the binding pocket of Poly28-PN. From this library, in some embodiments, yeast display platforms and FACS analyses were employed to isolate the constructs that exhibited the highest binding to DH270UCA3. These constructs were isolated through recombinant protein expression, and assessed for binding affinity to DH270.6-related antibodies using ELISA and SPR assays. Through quantitative measurements derived from such techniques, it was determined that these second-generation constructs (Poly281-PN and Poly283-PN) can bind DH270UCA3 at least five times better than Poly28-PN. It is understood that additional steps can be employed to generate primocalin constructs with KD values in the desired nanomolar-range to DH270UCA3 and related antibodies, these approaches are effective strategies for deriving immunogens that have both broad recognition and high affinity to target BCRs.

[0055] Step 3 in the process, in some embodiments, is testing the constructs for binding target antibodies using methods such as FACS and Surface Plasmon Resonance.

[0056] Step 4 in the process, in some embodiments, is testing the constructs for antibody binding and immune responses in cell lines and in mice.

[0057] Step 5 in the process, in some embodiments, is to take the information about the mutations and binding from steps 1-4 and develop a library of antibody target variants that contain all the single amino acid mutations in the CDR H3 loop.Sequences

[0058] Primocalin amino acid sequences obtained using the methods disclosed herein are shown below. In some embodiments, sequences that are 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequences below are disclosed.

[0059] >Yl_pdb (SEQ ID NO: 1)

[0060] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYAT IYELKEDKSYNVTSVLFADNKCTYWINTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVS TNYNQHAMVFFKMVTMNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0061] >Y2 _pdb (SEQ ID NO: 2)

[0062] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYAT IYELKEDKSYNVTSVLFADNKCTYWINTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVS TNYNQHAMVFFKMVNFNRESFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0063] >Y3_pdbN (SEQ ID NO: 3)

[0064] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFAT IYELKEDKSYNVTSVGFPDKACMYRIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVS TNYNQHAMVFFKKVSGNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0065] >Y4_pdbN (SEQ ID NO: 4)

[0066] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYAT IYELKEDKSYNVTSVLFADNKCTYWINTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVS TNYNQHAMVFFKMVTFNREWFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0067] >Y5_pdbN (SEQ ID NO: 5)

[0068] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYAT IYELKEDKSYNVTSVLFADNKCTYWIQTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVS TNYNQHAMVFFKMVDPNRESFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0069] >Y6 (SEQ ID NO: 6)

[0070] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYATIYELKEDKSYNVTSVLFADNKCTYWINTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVSTNYNQHAMVFFKMVDLNREKFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGLE

[0071] >Y7 (SEQ ID NO: 7)

[0072] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYATIYELKEDKSYNVTSVLFADNKCTYWIPTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVSTNYNQHAMVFFKMVTFNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGLE

[0073] >Y8_yeastN (SEQ ID NO: 8)

[0074] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYATIYELKEDKSYNVTSVLFADNKCTYWIPTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVSTNYNQHAMVFFKMVTLNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0075] >Y9_yeastN (SEQ ID NO: 9)

[0076] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTSVGFPDKACMYLIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSGNAEYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0077] >Y10_yeastN (SEQ ID NO: 10)

[0078] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYATIYELKEDKSYNVTSVLFADNKCTYWINTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVSTNYNQHAMVFFKMVSFNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGLE

[0079] >Y1 l_yeastN (SEQ ID NO: 11)

[0080] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYATIYELKEDKSYNVTSVLFADNKCTYWINTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVSTNYNQHAMVFFKMVSMNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPV PIDQCIDGLE

[0081] >Y12_yeastN (SEQ ID NO: 12)

[0082] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMYATIYELKEDKSYNVTSVLFADNKCTYWINTFVPGSQPGEFTLGNIKSYPGLTSFLVRVVSTNYNQHAMVFFKMVTLNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPV PIDQCIDGLE

[0083] >Y13_\ eastN (SEQ ID NO: 13)

[0084] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTSVGFPDKACMYLIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKMVESNREQFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPV PIDQCIDGLE

[0085] >Y14_veastN (SEQ ID NO: 14)

[0086] DLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTSVGFPDKACMYLIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSGNAEYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPV PIDQCIDGLE

[0087] >PC_POLY281_PN (SEQ ID NO: 15)

[0088] QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKMVTRNREEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0089] >POLY281_SH (SEQ ID NO: 16)

[0090] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKMVTRNREEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0091] >POLY310 (SEQ ID NO: 17)

[0092] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKGVRDNPEEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0093] >POLY311 (SEQ ID NO: 18)

[0094] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKGVRDNPELFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0095] >POLY312 (SEQ ID NO: 19)

[0096] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKNVRDNPEEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0097] >POLY313 (SEQ ID NO: 20)

[0098] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKGVHENPELFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0099] >POLY315 (SEQ ID NO: 21)

[0100] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKEVTYNEERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0101] >POLY316 (SEQ ID NO: 22)

[0102] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKVVEDNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0103] >POLY317 (SEQ ID NO: 23)

[0104] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKYVELNQERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0105] >POLY321 (SEQ ID NO: 24)

[0106] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKEDKSYNVTTVGFPDKACMYVTSTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKMVTRNREEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPV PIDQCIDG

[0107] >POLY322 (SEQ ID NO: 25)

[0108] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFADIYELKEDKSYNVTSVGFPDKACMYIIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKMVTRNREEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPI DQCIDG

[0109] >POLY323 (SEQ ID NO: 26)

[0110] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFAEIYELKEDKSYNVTSVGFPDKACMYIIKTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKMVTRNREEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPI DQCIDG

[0111] >PQLY401 (SEQ ID NO: 27)

[0112] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKEDKSYNVTTVGFPDKACMYVISTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKGVRDNPEEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0113] >POLY402 (SEQ ID NO: 28)

[0114] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKEDKSYNVTTVGFPDKACMYVISTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKGVRDNPELFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0115] >POLY403 (SEQ ID NO: 29)

[0116] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKEDKSYNVTTVGFPDKACMYVISTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKVVEDNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0117] >POLY404 (SEQ ID NO: 30)

[0118] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKEDKSYNVTTVGFPDKACMYVTSTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKNVRDNPEEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0119] >POLY405 (SEQ ID NO: 31)

[0120] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFADIYELKEDKSYNVTSVGFPDKACMYIIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKGVRDNPEEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0121] >POLY310MM (SEQ ID NO: 32)

[0122] LIPAPPLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLnEsKDPAKMFATIYELKnDsSYNVTSVGFPDKACMYEIRTFVPnSsPGEFTLGNIsSYPGLTSYLVRVVSTNYsQHAMVFFKMVTRNREEFLITLYGRTKnLTSELKENFInFSKSLGLPENHIVFPVPIDQCIDG

[0123] >POLY310MM_G1 (SEQ ID NO: 33)

[0124] LIPAPPLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKGVRDNPEEFLITLYGRTKnLTSELKENFInFSKSLGLPENHIVFPVPIDQCIDG

[0125] >POLY310MM_G2 (SEQ ID NO: 34)

[0126] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKGVRDNPEEFLITLYGRTKnLTSELKENFInFSKSLGLPnNsIVFPVPIDQCIDG

[0127] >POLY310MM_G3 (SEQ ID NO: 35)

[0128] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREnKsPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIsSDAGLTSYLVRVVSTNYsQHAMVFFKGVRDNPEEFLITLYGRTKnLTSELKENFInFSKSLGLPnNsIVFPVPIDQCIDG

[0129] >POLY316MM (SEQ ID NO: 36)

[0130] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVS TNYNQHAMVFFKVVEDNRERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0131] >POLY316MM G1 (SEQ ID NO: 37)

[0132] LIPAPPLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKVVEDNRERFLITLYGRTKnLTSELKENFInFSKSLGLPENHIVFPVPIDQCIDG

[0133] >POLY316MM_G2 (SEQ ID NO: 38)

[0134] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKVVEDNRERFLITLYGRTKnLTSELKENFInFSKSLGLPnNsIVFPVPIDQCIDG

[0135] >POLY316MM_G3 (SEQ ID NO: 39)

[0136] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREnKsPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIsSDAGLTSYLVRVVSTNYsQHAMVFFKVVEDNRERFLITLYGRTKnLTSELKENFInFSKSLGLPnNsIVFPVPIDQCID G

[0137] >POLY317MM (SEQ ID NO: 40)

[0138] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKEDKSYNVTDVGFPDKACMYVIRTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKYVELNQERFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVP IDQCIDG

[0139] >POLY317MM_G1 (SEQ ID NO: 41)

[0140] LIPAPPLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKYVELNQERFLITLYGRTKnLTSELKENFInFSKSLGLPENHIVFPVPIDQ CIDG

[0141] >POLY317MM G2 (SEQ ID NO: 42)

[0142] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREDKDPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKYVELNQERFLITLYGRTKnLTSELKENFInFSKSLGLPnNsIVFPVPIDQCIDG

[0143] >POLY317MM G3 (SEQ ID NO: 43)

[0144] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLREnKsPAKMFATIYELKnDsSYNVTDVGFPDKACMYVIRTFVPnSsPGEFTLGNIsSDAGLTSYLVRVVSTNYsQHAMVFFKYVELNQERFLITLYGRTKnLTSELKENFInFSKSLGLPnNsIVFPVPIDQCID G

[0145] >POLY401MM (SEQ ID NO: 44)

[0146] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKEDKSYNVTTVGFPDKACMYVISTFVPGSQPGEFTLGNIKSDAGLTSYLVRVVSTNYNQHAMVFFKGVRDNPEEFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0147] >POLY401MM_G1 (SEQ ID NO: 45)

[0148] LIPAPPLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKnDsSYNVTTVGFPDKACMYVISTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKGVRDNPEEFLITLYGRTKnLTSELKENFInFSKSLGLPENHIVFPVPIDQCIDG

[0149] >POLY401MM_G2 (SEQ ID NO: 46)

[0150] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAVLRKDKDPAKMFAAIYELKnDsSYNVTTVGFPDKACMYVISTFVPnSsPGEFTLGNIKSDAGLTSYLVRVVSTNYsQHAMVFFKGVRDNPEEFLITLYGRTKnLTSELKENFInFSKSLGLPnNsIVFPVPIDQCIDG

[0151] >Y524MM (SEQ ID NO: 47)

[0152] LIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIYRTDADPQKMVATIYELKEDKSYNVTIVLFADNKCTYHIETFVPGSQPGEFTLGNIKSDEGLTSFLVRVVSTNYNQHAMVFFKMVDPNRESFLITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPI DQCIDG

[0153] >Y524MM_G1 (SEQ ID NO: 48)

[0154] LIPAPPLSKVPLQQNFsDNQFQGKWYVVGLAGNAIYRTDADPQKMVATIYELKnDsSYNVTIVLFADNKCTYHIETFVPnSsPGEFTLGNIsSDEGLTSFLVRVVSTNYsQHAMVFFKMVDPNRESFLITLYGRTKnLTSELKENFInFSKSLGLPENHIVFPVPIDQCID G

[0155] >Y524MM_G2 (SEQ ID NO: 49)

[0156] LIPAPnLSKVPLQQNFsDNQFQGKWYVVGLAGNAIYRTDADPQKMVATIYELKnDsSYNVTIVLFADNKCTYHIETFVPnSsPGEFTLGNIsSDEGLTSFLVRVVSTNYsQHAMVFFKMVDPNRESFLITLYGRTKnLTSELKENFInFSKSLGLPENHIVFPVPIDQCID G

[0157] >MM_Poly28_GlSpyPaN (SEQ ID NO: 50)

[0158] TGHHHHHHENLYFQGAKFVAAWTLKAAAQDSTSDLIPAPPLSKVPLQQNFSDNQFQGKWYVVGLAGNAVLNESKDPAKMFATIYELKNDSSYNVTSVGFPDKACMYEIRTFVPNSSPGEFTLGNISSYPGLTSYLVRVVSTNYSQHAMVFFKMVTRNREEFLITLYGRTKNLTSELKENFINFSKSLGLPENHIVFPVPIDQCIDGSGESGRGVPHIVMVDAYKRYKG

[0159] >MM_Poly28_GlSpyPaC (SEQ ID NO: 51)

[0160] TGHHHHHHENLYFQGQDSTSDLIPAPPLSKVPLQQNFSDNQFQGKWYVVGLAGNAVLNESKDPAKMFATIYELKNDSSYNVTSVGFPDKACMYEIRTFVPNSSPGEFTLGNISSYPGLTSYLVRVVSTNYSQHAMVFFKMVTRNREEFLITLYGRTKNLTSELKENFINFSKSLGLPENHIVFPVPIDQCIDGAKFVAAWTLKAAASGESGRGVPHIVMVDA YKRYKG

[0161] >MM_Y513_GlSpyPaN (SEQ ID NO: 52)

[0162] TGHHHHHHENLYFQGAKFVAAWTLKAAAIPAPPLSKVPLQQNFSDNQFQGKWYVVGLAGNAIYNTSADPQKMYATIYELKNDSSYNVTMVLFADNKCTYHIQTFVPNSSPGEFTLGNISSDEGLTSFLVRVVSTNYSQHAMVFFKMVDPNRESFLITLYGRTKNLTSELKENFINFSKSLGLPENHIVFPVPIDQCIDGSGESGRGVPHIVMVDAYKRYK G

[0163] >MM_Y513_GlSpyPaC (SEQ ID NO: 53)

[0164] TGHHHHHHENLYFQGIPAPPLSKVPLQQNFSDNQFQGKWYVVGLAGNAIYNTSADPQKMYATIYELKNDSSYNVTMVLFADNKCTYHIQTFVPNSSPGEFTLGNISSDEGLTSFLVRVVSTNYSQHAMVFFKMVDPNRESFLITLYGRTKNLTSELKENFINFSKSLGLPENHIVFPVPTDQCIDGAKFVAAWTLKAAASGESGRGVPHIVMVDAYKRYKG

[0165] In some embodiments, disclosed here are nucleic acids that encode the polypeptides of SEQ ID NOs: 1-58, or nucleic acid sequences that are 90, 91, 92, 93. 94. 95. 96, 97, 98 or 99% identical thereto.

[0166] In certain aspects, the invention provides compositions for immunizations to induce lineages of broad neutralizing antibodies. In certain embodiments, there is some variance in the immunization regimen; in some embodiments, the selection of HIV-1 envelopes may be grouped in various combinations of primes and boosts, either as nucleic acids, proteins, or combinations thereof. In certain embodiments the compositions are pharmaceutical compositions which are therapeutic and / or immunogenic. In certain embodiments, the compositions comprise amounts of envelopes which are therapeutic and / or immunogenic.

[0167] In one aspect the invention provides a composition for a prime, for a boost, or for a prime and boost immunization regimen comprising any one of the envelopes described herein, or any combination thereof wherein the envelope is a prime or boost immunogen. In certain embodiments the composition for a prime boost immunization regimen comprises one or more envelopes described herein.

[0168] In certain embodiments, the compositions contemplate nucleic acid, as DNA and / or RNA, or recombinant protein immunogens either alone or in any combination. In certain embodiments, the methods contemplate genetic, as DNA and / or RNA, immunization either alone or in combination with recombinant envelope protein(s).

[0169] In some embodiments the antigens are nucleic acids, including but not limited to mRNAs which could be modified and / or unmodified. See US Pub 20180028645A1, US Pub 20170369532, US Pub 20090286852, US Pub 20130111615, US Pub 20130197068, US Pub 20130261172, US Pub 20150038558, US Pub 20160032316, US Pub 20170043037, US Pub 20170327842, each content is incorporated by reference in its entirety. mRNAs delivered in lipid nanoparticle (LNP) formulations have advantages over non-LNPs formulations. See US Pub 20180028645 Al.

[0170] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant.

[0171] In certain embodiments the induced immune response includes induction of antibodies, including but not limited to autologous and / or cross-reactive (broadly) neutralizing antibodies against HIV-1 envelope. Various assays that analyze whether an immunogenic composition induces an immune response, and the type of antibodies induced are known in the art and are also described herein.

[0172] In certain embodiments, where the nucleic acids are operably linked to a promoter and inserted in a vector, the vector is any suitable vector. Non-limiting examples include, VSV, replicating rAdenovirus type 4, MV A, Chimp adenovirus vectors, pox vectors, and the like. In certain embodiments, the nucleic acids are administered in NanoTaxi block polymer nanospheres. In certain embodiments, the composition and methods comprise an adjuvant. Non-limiting examples include, 3M052. AS01 B, AS01 E, gla / SE, alum, Poly I poly C (poly IC), polylC / long chain (LC) TLR agonists, TLR7 / 8 and 9 agonists, or a combination of TLR7 / 8 and TLR9 agonists (see Moody et al. (2014) J. Virol. March 2014 vol. 88 no. 6 3329- 3339), or any other adjuvant. Non-limiting examples of TLR7 / 8 agonist include TLR7 / 8 ligands, Gardiquimod, Imiquimod and R848 (resiquimod). A non-limiting embodiment of a combination of TLR7 / 8 and TLR9 agonist comprises R848 and oCpG in STS (see Moody et al. (2014) J. Virol. March 2014 vol. 88 no. 6 3329-3339). In non-limiting embodiments, the adjuvant is an LNP. See e.g., without limitation Shirai et al. “Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses'’ Vaccines 2020, 8, 433; doi: 10.3390 / vaccines8030433, published 3 August 2020.

[0173] In non-limiting embodiments, LNPs used as adjuvants for proteins or mRNA compositions are composed of an ionizable lipid, cholesterol, lipid conjugated with polyethylene glycol, and a helper lipid. Non-limiting embodiments include LNPs without polyethylene glycol.

[0174] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA. See Graham BS, Enama ME, Nason MC, Gordon IJ, Peel SA, et al. (2013) DNA Vaccine Delivered by a Needle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost ina Randomized Clinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies for delivery of nucleic acids, as DNA and / or RNA, so as to elicit immune response, both T-cell and humoral responses, are known in the art and are under developments. In certain embodiments, DNA is delivered as naked DNA. In certain embodiments, DNA is formulated for deliver}’ by a gene gun. In certain embodiments, DNA is administered by electroporation, or by a needle-free injection technology, for example but not limited to Biojector® device. In certain embodiments, the DNA is inserted in vectors. The DNA is delivered using a suitable vector for expression in mammalian cells. In certain embodiments the nucleic acids encoding the envelopes are optimized for expression. In certain embodiments DNA is optimized, e.g. codon optimized, for expression. In certain embodiments the nucleic acids are optimized for expression in vectors and / or in mammalian cells. In non-limiting embodiments these are bacterially derived vectors, adenovirus-based vectors, rAdenovirus (e.g. Barouch DH, et al. Nature Med. 16: 319-23, 2010), recombinant mycobacteria (e.g. rBCG or M smegmatis) (Yu, JS et al. Clinical Vaccine Immunol. 14: 886-093,2007; ibid 13: 1204-11,2006). and recombinant vaccinia type of vectors (Santra S. Nature Med. 16: 324-8, 2010), for example but not limited to ALVAC, replicating (Kibler KV et al., PLoS One 6: e25674, 2011 nov 9.) and non-replicating (Perreau M et al. J. virology 85: 9854-62, 2011) NYVAC, modified vaccinia Ankara (MV A)), adeno-associated virus, Venezuelan equine encephalitis (VEE) replicons, Herpes Simplex Virus vectors, and other suitable vectors.

[0175] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA or RNA in suitable formulations. Various technologies which contemplate using DNA or RNA, or may use complexes of nucleic acid molecules and other entities to be used in immunization. In certain embodiments, DNA or RNA is administered as nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer (amphiphilic block copolymer 704). See Cany et al., Journal of Hepatology 2011 vol. 54 j 115-121; Amaoty et al., Chapter 17 in Yves Bigot (ed.), Mobile Genetic Elements: Protocols and Genomic Applications, Methods in Molecular Biology, vol. 859, pp293-305 (2012); Arnaoty et al. (2013) Mol Genet Genomics. 2013 Aug;288(7-8):347-63. Nanocarrier technologies called Nanotaxi® for immunogenic macromolecules (DNA, RNA, Protein) delivery are under development. See for example technologies developed by incellart.

[0176] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the antibodies described herein. In certain embodiments, these are optionally formulated in lipid nanoparticles (LNPs). In certain embodiments, the mRNAs are modified. Modifications include without limitations modified ribonucleotides, poly-A tail, 5 'cap.

[0177] In certain aspects the invention provides nucleic acids encoding the antibodies or immunogens. In non-limiting embodiments, the nucleic acids are mRNA, modified or unmodified, suitable for use any use, e.g. but not limited to use as pharmaceutical compositions. In certain embodiments, the nucleic acids are formulated in lipid, such as but not limited to LNPs.

[0178] In some embodiments the immunogens are administered as nucleic acids, including but not limited to mRNAs which could be modified and / or unmodified. See US Pub 20180028645A1, US Pub 20090286852, US Pub 20130111615, US Pub 20130197068, US Pub 20130261172. US Pub 20150038558. US Pub 20160032316. US Pub 20170043037. US Pub 20170327842, US Patent 10,006,007, US Patent 9,371,511, US Patent 9,012,219, US Pub 20180265848, US Pub 20170327842, US Pub 20180344838A1 at least at paragraphs

[0260] -

[0281] , US Pub 20190153425 for non-limiting embodiments of chemical modifications, wherein each content is incorporated by reference in its entirety.

[0179] mRNAs delivered in LNP formulations have advantages over non-LNPs formulations. See US Pub 20180028645A1, US Pub 20190274968, US Pub 20180303925, wherein each content is incorporated by reference in its entirety7.

[0180] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant.

[0181] In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences ofinvention. In certain aspects the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of the invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector.

[0182] In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.

[0183] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. In some embodiments, the RNA molecule is encoded by one of the sequences described herein. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding any one of the polypeptide sequences of the sequences of the invention, or a variant thereof or a fragment thereof.

[0184] In some embodiments, a RNA molecule of the invention may have a 5' cap (e.g. but not limited to a 7-methylguanosine, 7mG(5')ppp(5')NlmpNp). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of an RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7- methylguanosine via a 5'-to-5' bridge. A RNA molecule may have a 3' poly -A tail. It may also include a poly -A polymerase recognition sequence (e.g. AAUAAA) near its 3' end. In some embodiments, a RNA molecule useful with the invention may be single-stranded. In some embodiments, a RNA molecule useful with the invention may comprise synthetic RNA.

[0185] In certain embodiments, the recombinant nucleic acid sequence is an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the envelope. Optimization can also improve transcription and / or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability7and codon optimization; addition of a kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequenceencoding a signal peptide; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).

[0186] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as recombinant proteins. Various methods for production and purification of recombinant proteins, including trimers such as but not limited to SOSIP based trimers, suitable for use in immunization are known in the art. In certain embodiments recombinant proteins are produced in CHO cells.

[0187] It is readily understood that the envelope glycoproteins referenced in various examples and figures comprise a signal / leader sequence. It is well known in the art that HIV- 1 envelope glycoprotein is a secretory protein with a signal or leader peptide sequence that is removed during processing and recombinant expression (without removal of the signal peptide, the protein is not secreted). See for example Li et al. Control of expression, glycosylation, and secretion of HIV-1 gp!20 by homologous and heterologous signal sequences. Virology 204(l):266-78 (1994) (“Li et al. 1994”), at first paragraph, and Li et al. Effects of inefficient cleavage of the signal sequence of HIV- 1 gp!20 on its association with calnexin, folding, and intracellular transport. PNAS 93:9606-9611 (1996) (“Li et al. 1996”), at 9609. Any suitable signal sequence could be used. In some embodiments the leader sequence is the endogenous leader sequence. Most of the gpl20 and gpl60 amino acid sequences include the endogenous leader sequence. In other non-limiting examples, the leader sequence is human Tissue Plasminogen Activator (TP A) sequence, human CD5 leader sequence (e g. MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO: 54)). Most of the chimeric designs include CD5 leader sequence. A skilled artisan appreciates that when used as immunogens, and for example when recombinantly produced, the amino acid sequences of these proteins do not comprise the leader peptide sequences.

[0188] The immunogenic envelopes can also be administered as a protein prime and / or boost alone or in combination with a variety of nucleic acid envelope primes (e.g., HIV -1 Envs delivered as DNA expressed in viral or bacterial vectors).

[0189] Dosing of proteins and nucleic acids are readily determined by a skilled artisan. A single dose of nucleic acid can range from a few nanograms (ng) to a few micrograms (pg) or milligram of a single immunogenic nucleic acid. Recombinant protein dose can range from afew micrograms to a few hundred micrograms, or milligrams of a single immunogenic polypeptide.

[0190] Administration: In certain embodiments, the compositions are formulated with appropriate carriers using known techniques to yield compositions suitable for various routes of administration. In certain embodiments the compositions are delivered via intramuscular (IM), via subcutaneous, via intravenous, via nasal, via mucosal routes, or any other suitable route of immunization.

[0191] In certain embodiments, the compositions are formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. The compositions can include an adjuvant, such as, for example but not limited to 3M052, alum, poly IC, MF-59 or other squalene-based adjuvant, ASOIB, or other liposomal based adjuvant suitable for protein or nucleic acid immunization. In certain embodiments, the adjuvant is GSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic using HBsAg as vaccine antigen (Leroux-Roels et al., IABS Conference, April 2013). In certain embodiments, TLR agonists are used as adjuvants. In other embodiment, adjuvants which break immune tolerance are included in the immunogenic compositions.

[0192] In certain embodiments, the compositions and methods comprise any suitable agent or immune modulation which could modulate mechanisms of host immune tolerance and release of the induced antibodies. In non-limiting embodiments modulation includes PD- 1 blockade; T regulatory' cell depletion; CD40L hyperstimulation; soluble antigen administration, wherein the soluble antigen is designed such that the soluble agent eliminates B cells targeting dominant epitopes, or a combination thereof. In certain embodiments, an immunomodulatory agent is administered in at time and in an amount sufficient for transient modulation of the subject's immune response so as to induce an immune response which comprises broad neutralizing antibodies against HIV-1 envelope. Non-limiting examples of such agents is any one of the agents described herein: e.g. chloroquine (CQ), PTP1B Inhibitor - CAS 765317-72-4 - Calbiochem or MSI 1436 clodronate or any other bisphosphonate; a Foxol inhibitor, e.g. 344355 Foxol Inhibitor, AS 1842856 - Calbiochem; Gleevac, anti-CD25 antibody, anti-CCR4 Ab, an agent which binds to a B cell receptor for a dominant HIV-1 envelope epitope, or any combination thereof. In non-limiting embodiments, the modulationincludes administering an anti-CTLA4 antibody, OX-40 agonists, or a combination thereof. Non-limiting examples are of CTLA-1 antibody are ipilimumab and tremelimumab. In certain embodiments, the methods comprise administering a second immunomodulatory agent, wherein the second and first immunomodulatory agents are different.Multimeric Nanoparticles

[0193] Presenting multiple copies of antigens to B cells has been a longstanding approach to improving B cell receptor recognition and antigen uptake (See Batista et al. EMBO J. 2000 Feb 15; 19(4): 513-520). The improved recognition of antigen is due to the avid interaction of multiple antigens with multiple B cell receptors on a single B cells, which results in clustering of B cells and stronger cell signaling. Furthermore, multimeric presentation improves antigen binding to mannose binding lectin which promotes antigen trafficking to B cell follicles. Self-assembling complexes comprising multiple copies of an antigen are one strategy of immunogen design approach for arraying multiple copies of an antigen for recognition by the B cell receptors on B cells (Kanekiyo, M., Wei, C.J., Yassine, H.M., McTamney, P.M., Boyington, J.C., Whittle, J.R., Rao, S.S., Kong, W.P., Wang, L., and Nabel, G.J. (2013). Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H INI antibodies. Nature 499, 102-106; Ueda, G., Antanasijevic, A.. Fallas, J.A., Sheffler. W.. Copps. J., Ellis, D., Hutchinson. G.B.. Moyer. A.. Yasmeen, A., Tsybovsky, Y„ et al. (2020). Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens. Elife).

[0194] In some instances, the gene of an antigen is fused via a linker / spacer to a gene of a protein which could self-assemble. Upon translation, a fusion protein is made that can selfassemble into a multimeric complex — also referred to as a nanoparticle displaying multiple copies of the antigen. In other instances, the protein antigen could be conjugated to the selfassembling protein via an enzy matic reaction, thereby forming a nanoparticle displaying multiple copies of the antigen. Non-limiting embodiments of enzymatic conjugation include without limitation sortase mediated conjugation. In some embodiments, linkers for use in any of the designs of the invention could be 2-50 amino acids long, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids long. Incertain embodiments, these linkers comprise glycine and serine amino acid in any suitable combination, and / or repeating units of combinations of glycine, serine and / or alanine.

[0195] Non-limiting embodiments of sortase linkers could be used so long as their position allows multimerization of the envelopes. In a non-limiting embodiment, a C- terminal tag is LPXTG (SEQ ID NO: 56), where X signifies any amino acid but most commonly Ala, Ser, Glu, or a N-terminal pentaglycine repeat tag is added to the envelope trimer gene. In a non-limiting embodiment, a C-terminal tag is LPXTGG (SEQ ID NO: 55), where X signifies any amino acid but most commonly Ala, Ser, Glu.

[0196] Another approach to multimerize expression constructs uses staphylococcus sortase A transpeptidase ligation to conjugate envelope trimers to cholesterol. The trimers can then be embedded into liposomes via the conjugated cholesterol. To conjugate the trimer to cholesterol either a C-terminal LPXTG (SEQ ID NO: 56) tag or a N-terminal pentaglycine repeat tag is added to the envelope trimer gene. Cholesterol is also synthesized with these two tags. Sortase A is then used to covalently bond the tagged envelope to the cholesterol. The sortase A-tagged trimer protein can also be used to conjugate the trimer to other peptides, proteins, or fluorescent labels.

[0197] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary' of Microbiology' and Molecular Biology' (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed.. R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary^ of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.Definitions

[0198] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any' number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. 28. 29. 30. 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0199] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0200] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0201] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%. 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0202] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0203] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.EXAMPLES

[0204] The Examples / Methods have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.EXAMPLE 1 Preparation of Library DNA and Yeast Transformation, Design and Synthesis of Library DNA

[0205] Based on Rosetta computational models of primocalin-antibody interactions, experimental libraries were developed that tested all combinations of amino acids at four sites deemed important for antibody binding (Figure 2A-B). With the yeast transformation protocol described below, 1-10 million individual colonies were obtained from transformation with a target library. Thus, a library can sample all the possible combinations of the 20 amino acids at a maximum of four primocalin sites in order to ensure adequate coverage (approximately ten times more) of the sequence space (204= 160,000).

[0206] To develop the library. 3FW4 and 3DSZ pdbs were originally tested with DH270UCA3 from 6UM5 structure. Each pdb was relaxed and then manually docked with the Ab. New pdb were generated that were run with NATAA (SEQ ID NO: 57) at all positions to repack sidechains with the test dock protocol (same as dock xml without intensive score function and filters). Based on appearance of these complexes, 3FW4 was structure chosen as basis for further modeling. Resfile was generated wherein every internal facing amino acid in the B-sheets as well as all loop residues were allowed to sample ALLAA (SEQ ID NO: 58). Thirty seven design locations across chain A (LCN2) were used: 5S, 6D, 7L, 40A. 411, 42L, 43R, 44E, 46K, 47D, 49Q, 68S, 70L, 72R, 73K, 74K, 75K. 77D, 79W. 81R. 100Y, 103L. 104T. 106Y, 108V. 123F, 125K. 127S, 128Q. 129N, I30R. 132Y, 134K.

[0207] <ROSETTASCRIPTS>

[0208] <SCOREFXNS>

[0209] < / SCOREFXNS>

[0210] <TASKOPERATIONS>

[0211] <InitializeFromCommandline name="ifcm" / >

[0212] <ReadResfile name = "rrf ' filename="UCA_3FW4.resfile" / >

[0213] <RestrictToRepacking name="r2p" / >

[0214] < / TASKOPERATIONS>

[0215] <FILTERS>

[0216] <Ddg name="ddg" confidence- ' 0" / >

[0217] < / FILTERS>

[0218] <MOVERS>

[0219] <Backrub name="bckrub" / >

[0220] <PackRotamersMover name="packer" task_operations="ifcm,rrf' / >

[0221] <Docking name- ' dock 1" fullatom="l" local_refine=" 1 " design="l" / >

[0222] <ParsedProtocol name="pack_dock">

[0223] <Add mover_name="dockl" / >

[0224] <Add mover_name="packer" / >

[0225] <Add mover_name="bckrub" / >

[0226] < / ParsedProtocol>

[0227] <LoopOver name="loop" mover name- 'pack dock" iterations- ' 10" / >

[0228] < / MOVERS>

[0229] <PROTOCOLS>

[0230] <Add mover_name="loop" / >

[0231] <Add filter name-1ddg" / >

[0232] < / PROTOCOLS>

[0233] < / ROSETTASCRIPTS>

[0234] These computationally designed primocalin library' variants were fused to Aga2p subunits and flanked with c-myc peptide epitope tags to allow for subsequent fluorescence- activated cell sorting (FACS) analyses (Figure 1). The Aga2p subunit is exported to the surface of the yeast cell where it binds to Agalp via disulfide bonds and thus becomes anchored to the cell wall. The c-myc epitope tag serves as a binding site for fluorescent antibodies used to quantify antibody-binding via FACS.

[0235] Aga2p-primocalin-c-mv DNA pools were constructed using a BioXp DNA synthesis system, then further amplified with polymerase chain reaction (PCR) using libraryspecific primers to isolate full-length library' DNA from other undesired secondary products. To do so, gradient polymerase chain reactions (PCRs) (IL forward primer, IL reverse primer, 0.5 L library DNA, 12.5 L Q5 Master Mix (New England Biolabs). 10 L DI H2O) were used to assess the optimal annealing temperatures (TM) (ranging from 55 °C to 67 °C) for amplification of the full-length library DNA strands. Fifty additional PCR reactions (IL foryvard primer, IL reverse primer, 0.5 L library DNA, 25 L Q5 Master Mix, 22.5 L DI H2O) were run following the identification of ideal reaction conditions (Tw = 64.7°), then pooledtogether for gel extraction (Thermo Fisher kit) to remove primers, nucleotides, salt, agarose, and other impurities. 12 g of library DNA was used for ethanol precipitation prior to yeast transformation.Library Transformation into 5*. cerevisiae

[0236] Library DNA was ethanol precipitated with BamHI. Sall, Nhel digested pCTCON2 vector (AddGene) in a 3: 1 ratio by mass (12 g of library' DNA, 4 g of pCTCON2) using DNA pellet paint (EMD Millipore) to further concentrate and de-salt the samples for effective transformation (Figure 1).

[0237] S. cerevisiae EBY100 cells were transformed with the precipitated DNA library and vector samples via electroporation (Figure 1), as previously described by Wittrup.19EBY100 cells were incubated for three days at 30 °C with shaking at 225 rpm prior to transformation. An aliquot of this culture was inoculated into 100 mL of yeast extract- peptone-dextrose (YPD) media (Teknova) after 72 hours at an optical density of 0.3 at 600 nm (ODeoo). The aliquoted culture was grown on a platform shaker at 30 °C and 225 rpm until OD6oowas approximately 1.6 (~6 hours). The cell pellet was washed twice with 50 mL DI H O. and once with an electroporation buffer (IM sorbitol, 1 mM CaCh) at room temperature; cells were collected by centrifugation at 3000 rpm for three minutes at 25°C to remove media. The yeast cells were conditioned by resuspension in a solution consisting of 20 mL 0. 1 M lithium acetate (Li AC) and 10 mM dithiothreitol (DTT) (Thermo Fisher), and shaking for 30 minutes at 30 °C and 225 rpm.

[0238] After incubation, cell samples were collected by centrifugation for three minutes at 3000 rpm and 4 °C. washed with 50 mL of ice-cold electroporation buffer, and incubated on ice. Previously pelleted library and vector DNA were resuspended in 20L of DI H2O, added to electrocompetent cells resuspended in 400 iL electroporation buffer, and transferred to pre-chilled BioRad electroporation cuvettes. Samples were iced for five minutes prior to electroporation (voltage = 2500 V; capacitance = 25 F. resistance = infinity). A time constant of 1.8 was recorded for the Poly28-PN library. Electroporated cells were immediately transferred into 8 mL of a 1 : 1 mix of 1 M sorbitol: YPD media and incubated on a platform shaker for 225 rpm at 30 °C for one hour. Cells were spun down and resuspended in 10 mL SDCAA media (Teknova) and 100X Penicillin-Streptomycin (Pen-Strep) (Thermo Fisher), then expanded for 72 hours on a shaker at 225 rpm and 30 °C. A sample of theculture was sequenced and analyzed to ensure efficient uptake of full-length library sequences. Five mL of transformed cells were passaged into a new solution of 45 mL of SDCAA media and 100X Pen-Strep every' 48 hours prior to the initial screening steps.EXAMPLE 2 FACS Screening and Sequence Analysis of Isolated Primocalins.Library Screening with FACS

[0239] High affinity library clones (and those with broad recognition of B-cells) were isolated using a series of FACS screening steps. Library transformants were first induced in a galactose medium (SGCAA) (Teknova) to promote Aga2p-primocalin-c- / m'c protein expression on the surface of yeast. The expression of the Aga2p-primocalin-c-mvc fusion on the cell surface of yeast transformants is governed by the inducible GALI promoter. When transformants are grown in a glucose medium (SDCAA), transcription of the GALI promoter is inhibited. Galactose, on the other hand, promotes the production and exportation of the two-subunit glycoprotein o-agglutinin and its attached primocalin sequence and c-myc epitope tag (Figure 1). Transformants were washed two times with 25 mL SGCAA (and collected with centrifugation for 3 minutes at 3000 rpm), then induced in 25 mL SGCAA at 110xl07cells / mL.

[0240] Following induction for 24-36 hours at 30 °C with shaking at 225 rpm, the appropriate volume corresponding to 210x107cells (concentration determined by OD ) was aliquoted from the induced sample and spun down at 12000 x g for 1 minute (this setting was used for all subsequent spin steps). The pelleted cells were washed twice with 1 mL of cold, sterile PBSA. Cells were then labeled with the appropriate concentration (ranging from 0. 1 nM to 1 / IM) of DH270UCA3 or related antibodies, brought up to a total volume of 200 qL with PBSA, resuspended, and incubated for one hour with shaking at 35 rpm and 4 °C. After incubation, cells were washed twice with PBSA, and resuspended with 1:100 monoclonal anti-c-myc-FITC-antibody (Thermo Fisher) and 1 :20 -mouse-PE-IgG-antibody (BioLegend) (secondary labeling reagents); labeled-cells were protected from light degradation with foil and incubated at 4 °C with shaking for 30 minutes. Cells were transferred to mini-collection tubes, then analyzed via flow cyto etry.

[0241] The monoclonal anti-c-m'c'-FITC-antibody interacts with the c-myc epitope attached to primocalin constructs, and will fluoresce if a primocalin-fusion construct isdisplayed on the surface of a yeast cell (Figure 1). Anti-mouse-PE IgG interacts with the heavy chain of DH270UCA3 IgGs, and will fluoresce if a primocalin construct is bound to a DH270UCA3 antibody (Figure 1). Transformants that display primocalin constructs that are able to effectively interact with the DH270UCA3 antibody will additionally exhibit -mouse- PE IgG binding (PE-positive or double-positive). Thus, FITC- and PE-double-positive cells were collected, expanded in SDCAA media (5 mL of culture into 45 mL of SDCAA every 48 hours), and re-screened through multiple rounds of enhanced selection steps. The highest affinity binders were isolated and designated for sequence analysis.

[0242] Note that a sample of only cells (no labeling reagents), and a sample of cells labeled with only 1 : 100 FITC were also isolated, washed, and incubated at all steps delineated above, and served as controls for determining FITC and PE positive cells. Sequence Analysis of Isolated Library' Clones

[0243] DNA from high-affinity primocalin constructs was isolated via yeast miniprep (Zymoprep), and transformed into NEB5a E. coli. Forty-eight colonies were selected to be analyzed by Sanger Sequencing.EXAMPLE 3 Protein Expression and Purification of Primocalin Constructs.E. coll Protein Expression

[0244] cDNA sequences of isolated clones, as determined by sequence analyses, were synthesized commercially (Genescript) and cloned into E. coli pET29b (Novagen) expression vectors. These vectors feature a T7 RNA polymerase promoter gene directly upstream of the gene of interest; thus, T7 RNA polymerase drives expression of these cDNA clones. Notably, the T7 polymerase gene is transcribed only in the presence of isopropyl (3-d- 1- thiogalactopyranoside (IPTG), which releases lactose (lac) repression, and allows for expression of the protein. The pET29b expression vectors also contain a N-terminal 6xHistidine tag positioned in the same open reading frame (ORF) as the gene of interest, and a cleavage site for a sequence-specific protease to allow for removal of the tag following purification.

[0245] Each sample of plasmid DNA was resuspended to a concentration of 0. 1 pg / pL, and 1 pL was transferred to 25 pL of BL-21(DE3) cells. Cells were incubated on ice for 30 minutes, heat shocked at 42 °C for 30 seconds to promote uptake of the plasmid, thenincubated on ice for another 10 minutes. 500 pL of room temperature SOC outgrowth media (Thermo Fisher) was added to each sample, and then incubated at 37 °C for 45 minutes with shaking at 225 rpm. Cells were transferred to a rich medium of 5 mL lysogenic broth (LB) with kanamycin and 1-2% glucose. The presence of glucose is crucial in reducing expression of native E. coli proteins, and the "‘leaking,” or loss, of expression plasmids into the media.20Samples were incubated overnight at 37 °C with shaking at 225 rpm.

[0246] One mL of each cell culture was transferred to 50 mL of terrific broth (TB) (consisting of tryptone, yeast, and sodium chloride) with kanamycin. Cell cultures were induced with 25 pL IPTG after four hours of growth at 37 °C with shaking at 250 rpm (until optical density was in the range of approximately 0.5-0.6), at 16 °C. The samples continued to be properly aerated with shaking at 250 rpm for 16 hours following induction.Protein Purification

[0247] Following bacterial expression of the primocalin proteins of interest, the cells were harvested by spinning down at 4000 rpm for 20 minutes; resulting cell pellets were incubated at -80 °C for an hour. Cells were then lysed through re-suspension with B-PER Bacterial Protein Extraction Reagent (5 mL / 1 g cells) (Thermo Fisher). The lysing process was facilitated by shaking at 75 rpm for 30-45 minutes at room temperature. Protein pellets were collected by spinning down at 9370 rpm at 4 °C for 30 minutes.

[0248] Target constructs were first purified by immobilized metal affinity chromatography (IMAC) with sodium phosphate buffers on a nickel (Ni2+) resin. This step took advantage of the 6xHistidine tags attached to the primocalin proteins of interest by isolation via nickel-histidine interactions. The nickel resin was prepared by washing Ni-NTA beads (QiaGen) with 5 column volumes of DI H2O and 50 column volumes of lysis buffer (20 mM NaFhPCh, 500 mM NaCl, 2% glycerol, 10 mM imidazole). The processed resin was resuspended with the protein pellet, and incubated with shaking at 4 °C for 60 minutes at 35 rpm. The resulting sample was spun down at 3000 rpm for three minutes, re-suspended with the Ni-NTA beads in an elution column, and washed with 30-50 column volumes of endotoxin-free wash buffer (20 mM NaFLPCb, 500 mM NaCl, 2% glycerol, 30 mM imidazole). The protein constructs of interest were eluted with 1-3 mL of an endotoxin-free elution buffer (20 mM NaFEPCh, 500 mM NaCl, 2% glycerol, 250 mM imidazole, pH 8.0). The elution buffer contains excess imidazole, which competes with the constructs of interestfor interactions with the nickel resin, thus releasing the primocalin proteins constructed into the collection tube.

[0249] An additional purification step was performed using size-exclusion chromatography (SEC) with phosphate SEC buffer (20 mM NafEPCh, 150 mM NaCl) to ensure complete exclusion of native proteins.20Samples were concentrated to <1 mL byspinning at 4000 rpm and 4 °C, then loaded into an AKTA pure protein purification system (Cytiva). Samples were analyzed by2sodium dodecyl sulfate -poly acry lamide gel electrophoresis (SDS-PAGE; Figure 5A). The protein quantity was determined spectrophotochemically by measuring absorbance at 280 nm using Nanodrop 2000 (ThermoFisher). The yields were approximately 50 mg / L of culture for each sample of primocalin construct isolatedEXAMPLE 4 Affinity Assessment Techniques for Primocalin Constructs.

[0250] Two different techniques were used to assess the affinity of expressed primocalin protein constructs to DH270UCA3 and related antibodies.Enzyme-Linked Immunosorbent Assay (ELISA)

[0251] ELISA analyses were conducted first, as a preliminary means to quantify interactions between isolated primocalin constructs and DH270 antibodies. Primocalin protein constructs were diluted in 0.1 M sodium bicarbonate (100 pg / mL-200 pg / mL) and coated (50 uL / well) on 96-well high-binding ELISA microplates (Costar 3700) at 4 °C overnight. Plates were washed one time with 100 pL of SuperWash (PBS supplemented with 0.5% Tween20) and patted dry . Two hundred microliters of SuperBlock with sodium azide (300 mL Goat Serum (Thermo Fisher), 10 mL Tween20, 80 mL 25X PBS, 20 mL aqueous 5% sodium azide, and 80 g Whey Protein (BiPro) in 2L DI H2O) were added to each well, and plates were incubated for 1 hour at room temperature. Following incubation, DH270UCA3 and related antibodies were diluted and titrated in SuperBlock with sodium azide (with starting concentrations of 100 pg / mL - 500 pg / mL, and dilution factors of two or three), and 50 pL of these diluted samples were added to the corresponding wells to initiate binding interactions with the primocalin constructs. Plates were incubated for 1 hour following the addition of target antibodies, then washed two times with 100 pL of SuperWash and patted dry.

[0252] Fifty microliters of a 1 : 15000 dilution of Goat anti Human IgG horseradish peroxidase (HRP) (Thermo Fisher) in SuperBlock without sodium azide (300 mL Goat Serum, 10 mL Tween20, 80 mL 25X PBS, and 80 g Whey Protein in 2L DI H O) solution was next added to each well, followed by a 1 hour incubation at room temperature. Goat anti Human IgG HRP is a secondary labeling reagent that binds to immunoglobulin G antibodies (such as DH270 related antibodies). Plates were next washed four times with 100 pL of SuperWash and patted dry after incubation. 50 pL of room temperature 3,3’ ,5,5’- tetramethylbenzidine (TMB) substrate (Thermo Fisher) - a tertiary binding agent that emits a blue coloration upon interaction with Goat anti-human IgG HRP) - was added to each well after the wash steps. Notably, if binding was observed between the primocalin constructs and DH270 antibodies, a reaction that formed a blue solution in the microplate wells would proceed, with deeper colors indicating a higher binding interaction. This reaction was stopped 5 minutes after adding the TMB substrate with an acid solution (IX HC1 solution), and then processed in a plate reader (BioTek) for the quantification of the binding interactions at 450 nm.Surface Plasmon Resonance (SPR) Analysis

[0253] Surface Plasmon Resonance (SPR) analysis was conducted for several samples. Two distinct antibody capture techniques were utilized: direct immobilization of the DH270UCA3 antibody on the sensor surface via amine-link coupling; and a-hlgG Fc capture, where an antibody specific for human Fc domains is immobilized on the sensor surface, and DH270UCA3 antibodies are captured onto the surface created by these Abs via their Fc regions. The direct mobilization method presents less artifacts on the sensor surface, eliminating the possibility that primocalin immunogens are interacting with the a-hlgG ab. However, with this technique, heterogeneity in the orientation by which DH270UCA3 covalently binds to the sensor surface is frequently observed. Such phenomena may limit the interactions of the primocalin constructs with the desired CDR H3 regions of the antibodies. With a-hlgG capture, this issue is resolved; however, there is a possibility- that the interactions observed are between the primocalin constructs and a-hlgG (and are non-specific to the antibody of interest). Both methods have advantages and disadvantages; hence, both were used as metrics to ensure validity- of results. Phosphate-buffered saline was used as the running buffer for both capture techniques.EXAMPLE 5 Selection of Primocalins with Broader Antibody Recognition.

[0254] From a previously derived library of primocalin constructs, a clone (Poly28-PN) that exhibited broad recognition to DH270UCA3 and several related antibodies was selected through alternative FACS screening steps.Engineering First-Generation Primocalin Constructs

[0255] To engineer primocalin immunogens that are capable of interacting with DH270UCA3, the sequence of a wildty pe lipocalin protein was computationally modified with Rosetta to model interactions with the antibody (Figures 2A-2B). Through these simulations, two hypothetical orientations by which the CDR H3 loop of the DH270UCA3 antibody could bind to the primocalin protein pocket were identified. On the backbones of the best computational models derived from both orientations, experimental libraries were developed that tested all possible combinations of amino acids at four sites judged to be important for antibody binding.

[0256] Immunogen libraries w ere screened on the surface of yeast for binding to DH270UCA3 using FACS. After multiple rounds of selection, a pool of primocalin clones displaying high binding interactions to lOnM DH270CUA3 w as isolated.Selection of a First-Generation "Polvsiiecific" Clone (Poly28-PN)

[0257] To generate primocalins that recognize not just the CDR H3 loop of DH270UCA3, but also those of related antibodies that may evolve to become HIV bnAbs from the natural immune repertoire, yeast transformants from the aforementioned pool were processed through alternative screening steps using DH270UCA3 chimeric antibodies where the CDR H3 loop was replaced by a natural CDR H3 loop. Notably, these CDR H3 loops exhibit D gene regions identical to that of DH270UCA3, but are different in the regions surrounding the D gene, namely the neighboring stochastically generated A-nucleotide regions. Due to the large sequence variations in the surrounding regions, primocalin clones that display effective interactions with multiple antibodies from the panel can interact primarily with the D gene of the CDR H3 loop.

[0258] Specifically, clones from the first-generation pool w ere analyzed by FACS for interactions with 37 related antibodies; a subset of these antibodies are displayed in Figure 3A. Transformants displayed above base-level binding to the following antibodies, in orderfrom highest number of binders to least: 65 la (10.6% binders), 713c (10.5%), 188 (8.7%). 650b (8.2%), 713d (0.7%), 650a (0.3%), GSGN28 (0.1%), GSGN21 (0.1 %), GSGN12 (0.1%), GSGN24 (0.1%), and GSGN13 (0.1%). An immunogen that is able to broadly recognize related antibodies by interacting specifically with the D gene region of the CDR H3 loop should exhibit binding to most, if not all. of these variants. However, screening strategies that involve testing all combinations of these antibodies to identify such a clone are both inefficient and expensive. Instead, four antibodies (651a, 713d, 650a, and GSGN28) with significant variations in the surrounding stochastic A-nucIcotidc regions (Figure 3 A) - while also displaying relatively high levels of binding when compared to other, similar DH270UC A3 -antibodies (Figure 3B) - were selected to conduct a simplified selection process. Ideally, due to the high sequence variations surrounding the D gene regions of their CDR H3 loops, screening with combinations of these four antibodies will be sufficient for isolating primocalin constructs with selective binding to the CDR H3 D gene.

[0259] Thus, two additional selection steps were conducted in succession. First, cells that exhibited positive binding to 651a, 713d, and 650a were screened for binding to the GSGN28 antibody (100 nM). GSGN28 exhibited the highest sequence variation relative to all three of the other antibodies, and was thus deemed the most effective in probing for constructs with high ’poly spec ficity" to multiple BCRs. The 651a (0.1%) and 713d (0.3%) positive cells displayed a higher number of GSGN28 binders than the 650a cells (<0. 1%) (Figure 3C), and were designated for an additional round of screening; to maximize the possibility' that a construct with broad recognition to multiple BCRs was present in the isolated pools, subsequent sorts advanced with the samples containing the highest amounts of GSGN28 binders. 651a / GSGN28 and 713d / GSGN28 positive cells were collected and expanded in SDCAA media for 5-7 days before proceeding to the next selection step.

[0260] For the final selection round, 651a / GSGN28 positive transformants were labeled with 713d (100 nM), and 713d / GSGN28 positive cells were labeled with 651a (100 nM) (Figure 3D). In essence, cells that exhibited binding at this step displayed primocalin constructs with high binding to DH270UCA3 and three other related BCRs (651a, 713d, GSGN28) with large sequence variation in the A-nucleotide regions surrounding the CDR H3 D gene, making them candidates for broad recognition of DH270UCA3-related BCRs. Theyeast clones remaining in culture after these selection rounds predominantly contained one sequence, named Poly28-PN.

[0261] At the four sites selected for variation, Poly28-PN exhibited considerable amino acid shifts from the original wildty pe lipocalin (Figure 3E). Two of these were changes from nonpolar to polar amino acids: at residue 67, there was a shift from arginine (polar) to proline (nonpolar); at residue 72, a shift from aspartic acid (polar) to methionine (nonpolar) was observed. Such adjustments were expected: surface amino acids of the primocalin binding pocket, which can interact favorably with surrounding aqueous solutions initially, are buried upon interaction with DH270-related antibodies. Other changes seemed to indicate a shift to smaller amino acids. At residue 65, glycine replaced leucine; both are nonpolar, but glycine is a smaller amino acid. Similarly, at position 74, a shift from tryptophan to glutamic acid was observed; try ptophan is significantly larger than glutamic acid. Smaller amino acids allow for more physical space in the primocalin binding pocket for CDR H3 loop interaction. Importantly, all amino acid shifts observed of Poly28-PN were also necessary for the construct to achieve broad recognition of 651a, 713d, and GSGN28, in addition to DH270UCA3.

[0262] Further optimization through new library' designs was based on the Poly28-PN template, described next. Poly28-PN was also later characterized following recombinant production in E. coli with surface plasmon resonance (SPR) analysis (Figure 5 E-F), as a baseline affinity metric for comparison to the enhanced constructs.EXAMPLE 6 Development of Second-Generation Libraries from Poly28-PN.

[0263] Poly28-PN was derived from a pool of primocalin constructs that displayed binding interactions to DH270UCA3 at a concentration of 10 nM. However, the FACS screening process does not confer a true 1 : 1 binding interaction betw een the primocalin and antibody epitopes, but rather reflects a measure of the avidity of the binding interaction, since labeling with bivalent antibodies and secondary reagents confounds the assessment of the binding strength of a singular binding site. Because avidity represents the total binding strength of all sites of interaction, it reports binding interaction values that are significantly more enhanced than the true 1 : 1 affinity between the binding pairs.

[0264] In order to achieve immunogen constructs with 1: 1 affinities in the nanomolar range, then, additional maturation can be facilitated through new library generation and additional FACS screening steps with lower antibody (DH270UCA3) concentrations. Thus, a second-generation I i bran- was constructed from Poly28-PN, with constructs derived from this library predicted to display both broad specificity to a range of DH270-related antibodies and higher affinity to DH270UCA3. Following computational engineering of the DNA library, S. cerevisiae yeast were transformed to display these new primocalin constructs on yeast for further optimization via high throughput screening.Poly28-PN Second-Generation Library generation

[0265] From Poly28-PN, second-generation libraries were constructed based on structural analysis and Rosetta computational modeling. Four new sites that were not sampled in the previous libraries were predicted to be important for antibody binding in silico. At these sites, all the combinations of the 20 amino acids were tested in primocalin variants characterized experimentally in a subsequent library.

[0266] These libraries were built experimentally for display on the surface of yeast. Oligonucleotides encoding the Poly28-PN-derived library were synthesized on a BioXp system. The Poly28-PN library oligos were amplified by PCR, at TM = 64.7. Following the successful expansion and purification of library DNA, . cerevisiae yeast were transformed. The size of the transformed li brary was 1. 110 xlO6(determined via serial dilutions on selection plates).EXAMPLE 7 Identification and Isolation of High Affinity Clones from Second-Generation, Library Designs Based on the Polv28-PN Construct.

[0267] After the successful transformation of the Poly28-PN library clones into yeast, a series of FACS screening steps were employed to isolate primocalin constructs with enhanced affinity to DH270UCA3 from the new library generated: high affinity clones are necessary’ to elicit immune response, especially when target BCRs are rare in the natural human repertoire. The DNA of the highest binding clones were sequenced following their isolation. Note that in future experiments, these higher affinity constructs should be screened with DH270UCA3 chimeric antibodies that have replaced CDR H3 loops of relatedantibodies (such as those in Figure 3) to ensure continued broad recognition of related, naturally-occurring BCRs.Isolation of High Binding Poly28-PN Second-Generation Primocalin Immunogens

[0268] The Poly28-PN library transformants were first screened for binding to 100 nM DH270UCA3 antibody using FACS (Figure 4A). Binders were collected, expanded in SDCAA for 5 days, then sorted with 1 nM DH270UCA3; the resulting highest binding clones were further collected and sorted with 0.1 nM DH270UCA3 (Figure 4B). Several constructs were isolated from this last selection step, indicating a promising improvement in affinity' when compared to the highest binding clones in the original library (yvhich only displayed binding at the ten-nanomolar-scale on the yeast display platform) (Figure 4C). The magnitude of improvement could not be accurately determined however, as, again, this system does not report true affinity measurements.Sequence Analysis

[0269] At the completion of the selection rounds, the DNA of the remaining clones was purified via yeast miniprep (Zymoprep), transformed into E. coli, and sent to Sanger sequencing. This allowed for the evaluation of the amino acid sequences of these new, enhanced constructs, specifically at the four sites selected for variation during computational design. Observations of dramatic shifts in the amino acids at these sites relative to the clones from the Poly28-PN template would demonstrate that more optimal amino acid combinations at the four selected positions of variation exist than in the previous templates, and would indicate that the new high binding constructs exhibit enhanced interactions to DH270UCA3 and related BCRs when expressed recombinantly in E. coli.

[0270] Notably, the new constructs demonstrated similar shifts in amino acids from the parent Poly28-PN sequence at the four sites selected for variation (Figure 4D). At position 190, almost all of the constructs substituted the original serine (S) amino acid for aspartic acid (D), thus replacing a polar amino acid with a larger, negatively charged one. At position 223, it was observed that almost all of the constructs replaced their original glutamic acid (E), which is negatively-charged, with hydrophobic amino acids such as valine (V) and isoleucine (I). More variation was seen at positions 286 and 289. At position 286, the original first- generation construct had a tyrosine (Y) amino acid, which is aromatic. Several derived constructs exhibit polar, charged amino acids at this position, such as aspartic acid (D) orlysine (K). At position 289, it was observed that the parent template featured a proline (P) amino acid. In several second-generation constructs, less-entropically restrictive amino acids, such as alanine (A), tyrosine (Y), and glutamic acid (E) were observed instead.

[0271] The consistent shifts in the amino acid sequences from the original parent templates indicate a transition to more optimal amino acid combinations at the four selected positions of variation. With this information, and with the enhanced binding observed on the yeast display platform, it was predicted that these new primocalin constructs exhibit higher affinity than the Poly28 constructs when isolated and expressed recombinantly in E. coli.

[0272] The four most frequently observed clones were selected for such characterization. Amino acid sequences at the four sites of variation of each of these clones (in order from position 190, 223, 286, and 289) are as follows: DVDA (Poly281-PN), DIDY (Poly282-PN), DYKE (Poly283-PN), DVTD (Poly283-PN) (Figure 4D).EXAMPLE 8 Affinity Assays of Primocalin Protein Constructs.Protein Expression and Purification.

[0273] The yeast display platform provides an efficient method to isolate the highest binding constructs from libraries consisting of over 1 million primocalin variants. However, the binding strengths of purified primocalins should be quantified to determine the true interaction strength of the immunogen with target antibodies in the absence of a cell platform.

[0274] Hence, Poly281-PN, Poly282-PN, Poly283-PN, and Poly284-PN were expressed recombinantly in E. coli, and purified with both immobilized metal affinity' chromatography and size exclusion chromatography. A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system (SDS-PAGE) confirmed that the protein constructs isolated were of the expected size of primocalin peptides (approximately 15 kDa) (Figure 5A). Binding interactions were then measured by ELISA and SPR techniques.Binding Analysis by ELISA

[0275] For each of the isolated Poly281 -PN, Poly282-PN, Poly 283-PN, and Poly284-PN constructs, binding was assessed to three different antibodies: 1) DH270UCA, the inferred DH270.6 unmutated common ancestor used to computationally' design the original primocalin constructs, and the standard antibody target used for all binding metrics in this study; 2) 188, a DH270 chimeric antibody with a substituted CDR H3 loop to assess broadbinding capabilities of the primocalin constructs; and 3) DH270UCA_Ala, a DH270 mutated antibody where aromatic residues located in the D gene of the CDR H3 loop were mutated to alanine, used as a negative control to ensure that engineered binding interactions are specific to the CDR H3 loop.

[0276] In an initial experiment, microplate wells were coated with 100 pg / mL of the relevant primocalin proteins, and the three DH270 related antibodies were added using a serial dilution with a starting concentration of 100 pg / mL and a dilution factor of three. Faint interactions were observed of Poly281-PN and Poly283-PN to DH270UCA3 and 188, but nearly no binding activity was detected for either Poly282-PN and Poly284-PN when compared to baseline noise. Thus, the assay was repeated for Poly281-PN and Poly283-PN, with higher concentrations of both the primocalin proteins (200 pg / mL) and antibodies (starting at 500 pg / mL, dilution factor of tw o).

[0277] With these increased concentrations, detectable levels of binding activity of Poly281-PN and Poly283-PN to DH270UCA3 and 188 mAbs were observed (Figure 5B-C). The similar interaction levels of the constructs to both DH270UCA3 and 188 indicate that these constructs have broad reactivity to antibodies of this type and that they recognize primarily the conserved D gene of the CDR H3 loop. However, additional assays need to be conducted with other DH270 chimeric antibodies with replaced CDR H3 loops to confirm this observation. Further, despite the comparable binding levels to both antibodies, these interactions are significantly weaker when compared to those with Y513, a primocalin construct used as a positive control and that has a different interaction mode with DH270UCA3, resulting in high affinity for DH270UCA3 and related antibodies (Figure 5B- C). For example, at an antibody concentration of 500 pg / mL, the binding response (optical density at 450 nm) of Y513 to DH270UCA3 is 2.45, while that of Poly281 -PN is 0.24, and that of Poly283-PN is 0.37; the binding response of Y513 to 188 is 0.54, while that of Poly281-PN is 0.25, and that of Poly283-PN is 0.27 (Figure 5B-C). On average, the interactions ofY513 to DH270UCA3 are approximately ten times stronger than ofPoly281- PN and Poly283-PN, and the interactions to 188 are roughly twice as favorable for Y513 than for Poly281-PN and Poly283-PN. Note that, as expected, no binding signal above the baseline activity was observed ofY513, Poly281-PN, and Poly283-PN to DH27UCA_Ala,indicating that all interactions were specific between the primocalin peptides and the DH270 antibodies (Figure 5D).

[0278] ELISA assays serve as an excellent analytical tool for quantifying antigenantibody interactions. However, as with the yeast display platform, bivalent antibodies and labeling reagents were added to a coated base of primocalins for these assays, which masked 1 : 1 interactions between individual primocalin and antibody binding epitopes. Hence, for the purposes of this study, ELISA assays were used as a benchmark to gauge differences in the magnitude of binding interactions between isolated primocalin constructs and antibodies of interest, and not as an absolute measure of affinity.Binding Analysis by SPR

[0279] To determine true 1 : 1 binding affinities between the engineered primocalins and target antibodies, surface plasmon resonance (SPR) technology was employed to assess realtime binding of Poly281-PN to DH270UCA3. Due to the similar activity profiles observed of Poly281-PN to 188, as well as of Poly283-PN to both antibodies in the ELISA assays, these sets of binding partners were not further characterized; future experiments, however, should be conducted to ensure that similar patterns are observed with SPR techniques.

[0280] With two SPR techniques used (direct immobilization and a-hlgG), bivalent antibodies and secondary reagents were initially tethered to the sensor, while primocalin constructs were injected over the resulting antibody -coated surface. This was, broadly speaking, the opposite workflow of what was employed via the yeast display platform and ELISA assays. Because the primocalin constructs are monomeric, the interaction between the binding pocket of an individual immunogen to a single interaction site on a DH270UCA3 antibody reflects true 1 : 1 binding. Thus, the true affinities, and an indication of the true binding strength of the primocalin constructs, were determined through these assays. In this study, the equilibrium dissociation constant, KD, served as the affinity metric, and was calculated with SPR analyses. KD describes both the likelihood a primocalin immunogen will stick to a DH270 antibody (the on-rate, or fy), and sustain such an interaction (the off-rate, or kd) (KD = kdlka). A lower KD indicates a faster on-rate, or a lower off-rate, and points to a higher affinity interaction.

[0281] When comparing the real-time binding profile of Poly281-PN to that of Y513, it was observed that while both appear to have similar, fast off-rates, Poly281-PN has asignificantly lower binding response to DH270UCA3 when injected over the antibody-loaded sensor chip surface (Figure 5E). When looking specifically at the and k, constants measured for both direct immobilization and a-hlgG Fc capture techniques, it was confirmed that while the dissociation constants for Poly281-PN and Y513 to DH270UCA3 were of the same magnitude, the association constant to DH270UCA3 was approximately ten times greater for Y513 than Poly 281 -PN with both capture mechanisms (Figure 5G-H). Given that the equilibrium dissociation constant (KD) is calculated as a ratio of off-rate to on-rate, it is approximately ten times lower for Y513 than Poly281-PN; hence, the binding strength for Y513 is ten times stronger to DH270UCA3 than Poly281-PN (Figure 5G-H). A similar discrepancy in binding strength between the two primocalin constructs to DH270UCA3 was observed with the ELISA experiments; consistent observations were recorded across both assays. Notably, while the off-rates do not contribute sizably to the discrepancy in binding affinity to DH270UCA3 between Poly281-PN and Y513, they significantly limit both primocalin constructs from achieving KDS in the desired nanomolar range. Thus, for Poly281- PN, future efforts to both increase the kaand decrease the kd constants are necessary.

[0282] Although the binding affinity' of Poly281-PN to DH270UCA3 was weaker than that of Y513, SPR data revealed that, when compared to the original Poly28-PN primocalin immunogen, binding strength of the new library construct increased by approximately a factor of 5 (Figure 5G-H) Closer assessments of both the kaand kd constants indicated that the computationally-determined enhancements and FACS screening steps both improved the association of the primocalin construct to the antibody and decreased the rate of dissociation following initial interactions (Figure 5G-H). These observations indicate that new library constructs generated from Poly28-PN. followed by additional rounds of FACS selection, could further enhance binding strength; repeated processes of this experimental cycle can eventually generate primocalins with KD values in the desired nanomolar range.EXAMPLE 9 Primocalin Nanoparticles.

[0283] Development of nanoparticles for immunization that display 60 copies of first- generation primocalins using the mi03 Spytag / Spy catcher platform. To achieve affinities similar to the previously isolated Y513, new sites important for the primocalin-antibody interaction were selected for variation, and additional rounds of high throughput library’screening to derive new constructs with even more enhanced binding strength. Additionally, to further enhance the affinity of all isolated primocalin constructs (including Y513) for target BCRs, we adopted the previously described SpyCatcher / Spytag mi60 nanoparticle technology. The SpyCatcher-SpyTag system creates an oligomeric immunogen that mimics a viral capsid and is thus able to both enhance the likelihood that immune mechanisms react to it, and amplify the intensity of the resulting responses. To create such a nanoparticle, SpyTags to the lipocalin immunogen proteins can be attached, which then covalently bond to a 60-mer molecule decorated with SpyCatcher domains. By mounting the designed primocalin immunogens on this platform, the apparent off-rates of these constructs can be improved by increasing the points of immunogen-antibody interaction, thus optimizing the KDconstants (and apparent affinities) of these lipocalin proteins (Figure 6).

[0284] Testing these nanoparticles in a DH270UCA3 Ramos B Cell line where binding to DH270UCA3 of the Y5-mi3 nanoparticles opened the Ca2+channels showing selectivity (Figure 7).EXAMPLE 10 Testing in Mouse Models with Diverse CDR H3 Loops.

[0285] Testing of first-generation primocalins in vivo was done in three different animal models: the DH270UCA3 KI mouse model and two different CDR H3 rearranging DH270UCA3 mouse models, the RV1 and RV2 models as shown (Figure 8A-C). The results demonstrate that first-generation primocalins:

[0286] a. recognize DH270 precursors with diverse CDR H3 loops in vitro.

[0287] b. robustly activate and expand DH270 UCA3 precursors in the KI model.

[0288] c. Engage a large number of DH270 precursors with diverse CDR H3 loops in the rearranging mouse models (Figure 9-10).EXAMPLE 11 Second- and Third-Generation Primocalins.

[0289] Development of second- and third-generation primocalins with improved affinity for DH270UCA3 and greater breadth of recognition of DH270 precursors containing more diverse CDR H3 loops (Figure 12-17).EXAMPLE 12

[0290] Deep scanning mutagenesis of DH270UCA3 CDR H3 libraries to determine in vitro the diversity of CDR H3 loops of DH270UCA3-like precursors that primocalins can recognize.EXAMPLE 13

[0291] Engineered DH270 germline-targeting SOSIP immunogen 10. 17DT.GSEXAMPLE 14

[0292] 10. 17DT.GS was tested in three mouse models: DH270UCA4 KI. DH270UCA3KI, and a rearranging DH270UCA3 model. We demonstrated that 10.17DT.GS robustly activates DH270 precursors in the DH270 UCA4 KI model and it leads to the acquisition of the G57R mutation in over 80% of activated DH270 UCA precursors. The ability to engage DH270 UCA4 and to promote the acquisition of the G57R mutation at such high frequencies are unique features of this germline targeting immunogen.EXAMPLE 15

[0293] Complete the in vivo characterization of 10. 17DT.GS and first-generation pnmocalin immunogens in relevant mouse models, by isolating and analyzing monoclonal antibodies from immunized animals and by characterizing the immune repertoire of vaccinated animals with next-generation sequencing. Second and third-generation primocalins will be similarly characterized in vivo. More broadly, the two key questions we will attempt to answer in the next year are: 1) what is the nature of the engaged precursors and how readily can they evolve into bnAbs and 2) what are appropriate boosting immunogens that can promote the evolution of precursors engaged by our germ line targeting immunogens into bnAbs. These questions will be answered using a combination of animal studies, immune repertoire analysis and in vitro characterization of isolated antibodies induced by vaccination.EXAMPLE 16

[0294] Figure 18 illustrates an example crystal structure of Y513 primocalin.

[0295] Figure 19 depicts example data showing that primocalins recognize antibodies with diverse CDR H3 loops. ( 19A) shows example structural data. ( 19B) shows example binding data.

[0296] Figure 20 depicts example binding data.

[0297] Figure 21 illustrates an example crystal structure of DH270UCA3 in complex with Poly 310.

[0298] Figure 22 depicts example immunization studies with primocalins.

[0299] Figure 23 depicts example data illustrating that primocalins can help select forCDR H3 loops specific for anti-HIV broadly neutralizing antibodies (bnAbs). The combination of a primocalin priming immunization and 10.17 boost is superior to 10.17 alone.

[0300] Figure 24 depicts example data for sequences of 10. 17DT (x4) elicited clones. The data show the diversity of the CDR H3 loop sequences generated.

[0301] Figure 25A-D. (25A-C) depict example data for sequences of Poly28 (x4) elicited clones. (25D) depicts example data for sequences of 10.17DT (x4) elicited clones. The data show the diversity of the CDR H3 loops sequences generated.

[0302] Figure 26 A-E. (25A-C) depict example data for sequences of Poly 28 (x2) + 10. 17DT (x2) elicited closes. (25D) depicts example data for sequences of 10. 17DT (x4) elicited clones. (25E) depicts example data for sequences of Poly28 (x4) elicited clones.

[0303] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0304] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0305] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.References

[0306] Bartlett, J. G., Redfield, R. R., & Pham, P. A. (2019). Antiretroviral therapy. Bartlett's Medical Management of HIV Infection, 175-262. https : / / doi. org / 10.1093 / med / 9780190924775.003.0004.

[0307] Schackman, B. R., Fleishman, J. A., Su, A. E , Berkowitz, B. K., Moore, R. D., Walensky, R. P., Becker, J. E., Voss, C., Paltiel, A. D., Weinstein, M. C., Freedberg, K. A., Gebo, K. A., & Losina, E. (2015). The lifetime medical cost savings from preventing FIIV in the United States. Medical Care, 53(4), 293-301. https : / / doi. org / 10.1097 / mlr.0000000000000308.

[0308] Kwong, P. D., & Mascola, J. R. (2018). HIV-1 vaccines based on antibody identification, B cell ontogeny, and epitope structure. Immunity, 48(5), 855-871. https: / / doi.Org / 10.1016 / j.immuni.2018.04.029.

[0309] Li, Y., O'Dell, S.. Walker, L. M., Wu, X , Guenaga, J., Feng, Y., Schmidt, S. D , McKee, K., Louder, M. K., Ledgerwood, J. E., Graham, B. S., Haynes, B. F., Burton, D. R., Wyatt, R. T., & Mascola, J. R. (2011). Mechanism of neutralization by the broadly neutralizing HIV- 1 monoclonal antibody VRC01. Journal of Virology, 85(17), 8954-8967. https: / / doi.org / 10. 1128 / jvi.00754-11.

[0310] Parsons, M. S., Kristensen, A. B., Selva, K. J., Lee, W. S., Amarasena, T., Esterbauer, R., Wheatley, A. K., Bavinton, B. R., Kelleher, A. D., Grulich, A. E., Khoury, G., Juno, J. A., & Kent, S. J. (2021). Protective efficacy of the anti-HIV broadly neutralizing antibody PGT121 in the context of semen exposure. EBioMedicine, 70, 103518. https : / / doi. org / 10.1016 / j . ebiom.2021.103518.

[0311] Bonsignori, M., Zhou, T., Sheng, Z., Chen, L., Gao, F., Joyce, M. G., Ozorowski, G., Chuang, G.-Y., Schramm, C. A., Wiehe, K., Alam, S. M., Bradley, T., Gladden, M. A., Hwang, K.-K., Iyengar, S., Kumar, A., Lu, X., Luo, K., Mangiapani, M. C., ... Haynes, B. F. (2016). Maturation pathway from germline to broad HIV-1 neutralizer of a CD4-mimic antibody. Cell, 165(2), 449-463. https: / / doi.Org / 10.1016 / j.cell.2016.02.022.

[0312] Haynes, B. F., Kelsoe, G., Harrison, S. C., & Kepler, T. B. (2012). B-cell-lineage immunogen design in vaccine development with HIV-1 as a case study. Nature Biotechnology. 30(5), 423-433. https: / / doi.org / 10.1038 / nbt.2197.

[0313] Bonsignori. M., Kreider. E. F.. Fera. D.. Meyerhoff. R. R., Bradley. T.. Wiehe. K., Alam, S. M., Aussedat, B., Walkowicz, W. E., Hwang, K.-K., Saunders, K. O., Zhang, R., Gladden, M. A., Monroe, A., Kumar, A., Xia, S.-M., Cooper, M., Louder, M. K., McKee, K., Haynes, B. F. (2017). Staged induction of HIV- 1 gly can-dependent broadly neutralizing antibodies. Science Translational Medicine. 9(381). https: / / doi.org / 10. ! 126 / scitr ansimed. aai7514.

[0314] Wu, T. T., Johnson, G., & Kabat, E. A. (1993). Length distribution of CDR H3 in antibodies. Proteins: Structure, Function, and Genetics, 16(1), 1-7. https : / / doi. org / 10.1002 / prot.340160102.

[0315] Abbott, R. K„ Lee, J. H., Menis, S„ Skog, P., Rossi, M., Ota, T., Kulp, D. W., Bhullar, D., Kalyuzhniy, O., Havenar-Daughton, C., Schief, W. R., Nemazee, D., & Crotty.S. (2018). Precursor frequency and affinity' determine B cell competitive fitness in germinalcenters, tested with germline-targeting HIV vaccine immunogens. Immunity, 48(\). https: / / doi.Org / 10.1016 / j.immuni.2017. l 1 .023.

[0316] Bassing, C. H., Swat, W., & Alt, F. W. (2002). The mechanism and regulation of chromosomal V(D) J Recombination. Cell. 109(2). https: / / doi.org / 10.1016 / s0092- 8674(02)00675-x.

[0317] Saunders, K. O., Wiehe, K, Tian, M., Acharya, P., Bradley, T., Alam, S. M., Go, E. P., Scearce, R., Sutherland, L., Henderson, R., Hsu, A. L., Borgnia, M. J., Chen, H., Lu, X., Wu, N. R„ Watts, B„ Jiang, C„ Easterhoff, D„ Cheng, H.-L., ... Haynes, B. F. (2019). Targeted selection of HIV-specific antibody mutations by engineering B cell maturation.Science, 366(6470). https: / / doi.org / 10.1126 / science.aay7199.

[0318] Briney, B. S., Willis, J. R., & Crowe, J. E. (2012). Human peripheral blood antibodies with long hcdr3s are established primarily at original recombination using a limited subset of germline genes. PLoS ONE, 7(5). https: / / doi.org / 10.1371 / joumal.pone.0036750.

[0319] Bassing, C. H., Swat, W., & Alt, F. W. (2002). The mechanism and regulation of chromosomal V(D)J Recombination. Cell, 109(2). https: / / doi.org / 10. 1016 / s0092-8674(02)00675-x.

[0320] Schiefner, A., & Skerra, A. (2015). The menagerie of human lipocalins: A natural protein scaffold for molecular recognition of physiological compounds. Accounts of Chemical Research, 48( ), 976-985. https: / / doi.org / 10.1021 / ar5003973.

[0321] Swanson, O., Beem, J. M., Rhodes, B., Wang, A., Barr, M., Chen, H., Parks, R., Saunders, K. O., Haynes, B. F., Wiehe, K., & Azoitei, M. L. (2021). Identification of CDR H3 loops in the naive B cell receptor repertoire that can be engaged by candidate immunogens, https: / / doi.org / 10.! 101 / 2021 .12.18.473225.

[0322] Leaver-Fay, A., Tyka, M., Lewis, S. M., Lange, O. F., Thompson, J., Jacak, R., Kaufman, K. W.. Renfrew, P. D., Smith, C. A., Sheffler, W., Davis, I. W., Cooper, S., Treuille, A., Mandell, D. J., Richter. F., Ban, Y.-E. A., Fleishman, S. J., Com, J. E., Kim, D. E., ... Bradley, P. (201 1). ROSETTA3. Computer Methods, Part C, 545-574. https: / / doi.org / 10.1016 / b978-0-12-381270-4.00019-6.

[0323] Boder, E. T., & Wittrup, K. D. (1997). Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnology, 15(6), 553-557. https : / / doi. org / 10. 1038 / nbt0697 -553.

[0324] Chao, G., Lau, W. L., Hackel, B. J., Sazinsky, S. L., Lippow, S. M.. & Wittrup, K. D. (2006). Isolating and engineering human antibodies using yeast surface display. Nature Protocols, 1(2), 755-768. https: / / doi.org / 10.1038 / nprot.2006.94.

[0325] Graslund, S., Nordlund, P., Weigelt, J., Hallberg, M., Bray, J., Gileadi, O., Knapp,S., Oppermann, U., Arrowsmith, C., Hui, R., Ming, J., dhe-Paganon, S., Park, H.-won, & Savchenko. A. (2008). Protein production and purification. Nature Methods, 5(2), 135-146. https: / / doi.org / 10.1038 / nmeth.f202.

[0326] Zhao, H., Gorshkova, I. I., Fu, G. L., & Schuck, P. (2013). A comparison of binding surfaces for SPR biosensing using an antibody-antigen system and affinity distribution analysis. Methods, 59(3), 328-335. https: / / doi.Org / 10.1016 / j.ymeth.2012.12.007.

[0327] Azoitei, M. L., Ban, Y. A.. Kalyuzhny, O.. Guenaga, J., Schroeter, A., Porter. J., Wyatt, R., & Schief, W. R. (2014). Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5. Proteins: Structure, Function, and Bioinformatics , <82(10), 2770-2782. https: / / doi.org / 10.1002 / prot.24641.

[0328] Joyce, M. G.. Wheatley, A. K.. Thomas, P. V., Chuang, G.-Y., Soto, C., Bailer. R.T., Druz. A.. Georgiev, I. S.. Gillespie, R. A., Kanekiyo, M., Kong. W.-P., Leung, K., Narpala, S. N., Prabhakaran, M. S., Yang, E. S., Zhang, B., Zhang, Y., Asokan, M., Boyington, J. C., ... McDermott, A. B. (2016). Vaccine-induced antibodies that neutralize group 1 and Group 2 Influenza A viruses. Cell, 166(3), 609-623. https : / / doi. org / 10. 1016 / j . cell.2016.06.043.

[0329] Stadlbauer, D., Zhu, X., McMahon, M., Turner, J. S., Wohlbold, T. J., Schmitz, A. J., Strohmeier, S., Yu, W., Nachbagauer, R., Mudd, P. A., Wilson, I. A., Ellebedy, A. H., & Krammer, F. (2019). Broadly protective human antibodies that target the active site of influenza virus neuraminidase. Science, 366(6464), 499-504. https: / / doi.org / 10. ! 126 / science.aay0678.

[0330] Li, D., Edwards, R. J., Manne, K., Martinez, D. R., Schafer, A., Alam, S. M., Wiehe, K., Lu, X., Parks, R., Sutherland, L. L., Oguin, T. H., McDanal, C., Perez, L. G., Mansouri, K., Gobeil, S. M. C , Janowska, K.. Stalls, V., Kopp. M., Cai, F., ... Saunders, K.O. (2021). In vitro and in vivo functions of SARS-COV-2 infection-enhancing and neutralizing antibodies. Cell, 184(\6). https: / / doi.Org / 10.1016 / j.cell.2021.06.021.

[0331] Starr, T. N., Czudnochowski, N., Liu, Z., Zatta, F., Park, Y.-J., Addetia, A., Pinto, D., Beltramello, M., Hernandez, P., Greaney, A. J., Marzi, R., Glass, W. G., Zhang, I., Dingens, A. S.. Bowen, J. E., Tortorici, M. A., Walls, A. C., Wojcechowskyj, J. A., De Marco, A., ... Snell, G. (2021). SARS-COV-2 RBD antibodies that maximize breadth and resistance to escape. Nature, 597(7^74), 97-102. https: / / doi.org / 10.1038 / s41586-021-03807- 6.

[0332] Brune, K., Leneghan, D.. Brian, I. et al. Plug-and-Display: decoration of Virus- Like Particles via isopeptide bonds for modular immunization. Set Rep 6. 19234 (2016). doi . org / 10. 1038 / srep 19234.

Claims

Claims:

1. A method, comprising developing immunogens that elicit antibodies containing functional features of interest, such as long CDR H3 loops, and can be applied to develop immunogens that target elicitation of antibodies containing long CDR H3 loops against HIV, influenza or coronaviruses.

2. The method of claim 1, comprising modifying the amino acid sequence of a lipocalin protein such that the modified amino acid sequence binds specifically to an unmutated common ancestor (UCA) of a broadly neutralizing antibody (bnAb) lineage.

3. The method of claim 2, wherein the immunogens have high binding affinity to a CDR H3 loop of the bnAb.

4. The method of claim 3, wherein the immunogens additionally interact with regions of the CDR H3 loop of the bnAb that are conserved across multiple bnAb precursors.

5. The method of claim 4, wherein the UCA comprises DH270.

6. The method of claim 2, wherein a library of the modified amino acid sequence of the lipocalin protein are screened for specific binding to the UCA of the bnAb.

7. The method of claim 6, wherein clones from the library that bind the UCA of the bnAb are further optimized.

8. The method of claim 7, comprising screening the clones that bind the UCA of the bnAb for those that bind to bnAbs with CDR H3 loops replaced by CDR H3 loops of naturally-occurring BCRs with identical D-gene regions.

9. The method of claim 8, wherein the replacement CDR H3 loops have similar amino acid lengths to the CDR H3 loops which have been replaced, and / or prioritize contacts of the immunogens to amino acids in the CDR H3 loops encoded by the D gene.

10. A method for making pre-priming germline targeting immunogens, comprising: making a library of proteins designed to be bound by an antibody containing a long CDR H3 loop; and screening the library7for proteins to which the antibody binds.

11. A method for designing proteins in a library where in the proteins can be bound by an antibody, comprising: selecting an antibody having a long CDR H3 loop (>20 amino acids); and designing a library of proteins having amino acids to which the CDR H3 loop of the antibody can bind.

12. The method of claim 11, wherein the library of proteins is based on a lipocalin protein.

13. The method of claim 11. wherein the antibody comprises a B-cell receptor.

14. The method of claim 11, wherein the amino acids to which the CDR H3 loop of the antibody can bind are derived from a V3-loop and surrounding glycans on an HIV-1 spike protein.

15. The method of claim 11, wherein the library7of proteins prioritizes contacts with CDR H3 loops in the antibody.

16. An immunogen made by the method of any one of claims 1-15.

17. The immunogen of claim 16, wherein the immunogen is capable of activating a naive B cell to eventually produce a broadly -neutralizing antibody against HIV-1.

18. An immunogen having high binding affinity to a CDR H3 loop of a broadly neutralizing antibody (bnAb) specific for a virus, comprising SEQ ID NOs: 1-53 or sequences at least 90% identical thereto.

19. An immunogen, comprising SEQ ID NOs. 1-53 or sequences at least 90% identical thereto.

20. A nucleic acid encoding the immunogen of claim 18 or 19.

21. A nanoparticle comprising the immunogen of claims 18 or 19.

Images