Immunization schedules to elicit broadly-neutralizing antibodies against human immmunodeficiency virus (HIV)

Abstract

Immunization schedules to elicit the production of antibodies against the human immunodeficiency virus (HIV), including Tier 2 HIV, are described. The strategies include immunization utilizing engineered and multimerized HIV envelope glycoproteins and SOSIP trimers. When B cells effectively mature against the HIV virus, subjects can be protected against infection of HIV.

Description

F053-0192PCT / 23-207-WO-PCT IMMUNIZATION SCHEDULES TO ELICIT BROADLY-NEUTRALIZING ANTIBODIES AGAINST HUMAN IMMMUNODEFICIENCY VIRUS (HIV) CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 612,268 filed December 19, 2023, which is incorporated herein by reference in its entirety as if fully set forth herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AI138212 and AI104384 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3CI8508.XML. The file is 127,785 bytes, was created on December 10, 2024, and is being submitted electronically via Patent Center. FIELD OF THE DISCLOSURE

[0004] The current disclosure provides immunization schedules that elicit the production of broadly-neutralizing antibodies against the human immunodeficiency virus (HIV), including Tier 2 HIV. The schedules include administration of engineered and / or multimerized antigens based on HIV group M antigens (e.g., 426c, HxB2, or consensus M). Exemplary schedules can include administration of 3-5 antigens over 9-21 weeks, in protein or nucleic-acid based forms. BACKGROUND OF THE DISCLOSURE

[0005] Acquired Immunodeficiency Syndrome (AIDS) is characterized by immunosuppression that results in opportunistic infections and malignancies, wasting syndromes, and central nervous system degeneration. Destruction of CD4+ T-cells, which are critical to immune defense, is a major cause of the progressive immune dysfunction that is the hallmark of AIDS disease progression. The loss of CD4+ T cells seriously impairs the body's ability to fight most pathogens, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.

[0006] AIDS is caused by infection with the human immunodeficiency virus (HIV). HIV infection begins when a portion of the virus’ envelope protein (Env), gp120, binds to CD4 and otherF053-0192PCT / 23-207-WO-PCT receptors on the surface of an infected subject’s CD4+ T-cells and other immune system cells. The bound virus then fuses with the bound infected subject’s cell and reverse transcribes its RNA genome. The resulting viral DNA integrates into the infected subject’s cell’s genome and begins to produce new viral RNA, resulting in new viral proteins and virions. The virions leave the originally infected cell to infect new cells. This process kills the originally infected cell.

[0007] Antibodies are proteins that can provide protection against pathogens, such as viruses. In some instances, antibodies are protective because they bind to an invading pathogen and interfere with its normal function. For example, many protective antibodies bind to a portion of a virus that blocks its ability to bind to and / or enter an infected subject’s cells.

[0008] Vaccines are designed to increase the immunity of a subject against a particular pathogen by exposing subjects to an innocuous form or portion of the pathogen that will not lead to an active and / or on-going infection. Exposure to the form or portion of the pathogen stimulates B cells to produce antibodies against the targeted pathogen.

[0009] Each B cell expresses a unique antibody with unique specificity for a particular epitope on a protein. The unique antibody expressed by each B cell is generated randomly through genetic recombination. A germline (gl) B cell refers to an immature B cell before it has come into contact with its epitope. gl B cells express membrane-bound antibodies (also called B cell receptors or BCR). When a BCR encounters and binds its particular epitope, the B cell begins to rapidly proliferate and mature. During proliferation and maturation, the B cell’s antibody genes undergo somatic hypermutation, which serves to increase the affinity of the B cell’s antibody to its initial epitope. The increase in affinity of epitope binding that occurs during B cell maturation is required for effective protection against the pathogen. A single gl B cell is able to undergo dozens of cell divisions to create thousands of antibody-secreting B cells and memory B cells expressing the same antibody, or a related antibody that has been mutated to improve binding to the pathogen.

[0010] For decades, researchers have been trying to develop a vaccine that can induce B cells to produce antibodies that are effective to protect against HIV. VRC01-class antibodies are among the most broad and potent neutralizing antibodies known against HIV. Balazs et al., Nature. 2012;481(7379):81-4; Balazs et al., Nature Medicine.2014;20(3):296-300; Shingai et al., Journal of Experimental Medicine.2014;211(10):2061-74. VRC01-class antibodies are important targets for an HIV-1 vaccine because they are among the most broad and potent neutralizing antibodies known; their passive administration to non-human primates prior to SHIV-exposure or their active expression in humanized mice prior to HIV-1 exposure leads to protection from infection (in Balazs et al., Nature 481, 81-84, (2012). Balazs et al., (Nat Med 20, 296-300, (2014)), Shingai et al., J Exp Med 211, 2061-2074, (2014), and two phase 3 clinical trials (HVTN 703 / 704) demonstratedF053-0192PCT / 23-207-WO-PCT that a member of that antibody class (mAb VRC01) prevents HIV-1 acquisition from VRC01- sensitive tier 2 viruses (in Corey et al., N Engl J Med 384, 1003-1014, (2021)). The development of an immunization protocol that will reproducibly elicit VRC01-like broadly neutralizing antibodies (bNAbs) will be of high clinical significance to the development of such a vaccine.

[0011] Engineered HIV Env proteins that bind to germline B cell receptors (glBCRs) (including glVRC01 BCRs) and activate B cells have been developed. McGuire et al., J Exp Med 210(4), 655-663 (2013) and WO2016 / 154422 describe an engineered Env protein (the ‘426c core’) that binds and activates the inferred gl forms of BCR, including diverse VRC01-class bNAbs. This protein was derived from the Clade C virus 426, which was isolated from an HIV patient 90 days post-infection. Jardine et al., Science, 340 (6133), 711-716 (2013) and WO2016 / 205704 describe “engineered outer domain” or “eOD” Env proteins that bind glVRC01 BCR. Medina-Ramierz et al., J Exp Med 214(9), 2573-2590 (2017) describes the engineered BG505 SOSIP.664 glycoprotein, BG505 SOSIP.v4.1-GT1 trimer. While immunization with these engineered HIV Env proteins alone stimulate glBCR that can lead to broadly-neutralizing antibodies against HIV, the stimulated glBCR do not sufficiently mature to create broadly-neutralizing antibodies.

[0012] One of the important reasons for the lack of complete success to date is due to the fact that the appropriate maturation of glBCRs (including VRC01 BCRs) requires that somatic hypermutations during maturation effectively circumvent steric binding constraints on HIV’s Env protein. For example, HIV-1 has evolved to avoid detection by glVRC01 B cells. HIV-1 avoids detection by glVRC01 B cells through the presence of specific N-linked glycosylation sites (NLGS) within the gp120 protein. One such NLGS is found at position 276 of the Env. As a consequence, recombinant Env proteins derived from diverse HIV-1 isolates are ineffective in binding to and stimulating B cells that express the glBCR forms of VRC01-class bNAbs. Targeted disruption of certain conserved NLGS, however, permits binding and activation gl B cell lines expressing BCRs of two clonally-related VRC01-class bNAbs, VRC01 and NIH45-46. These two BCRs represent a small subset of potential VRC01-class antibody progenitors. Thus, designing immunogens capable of recognizing a larger group of glBCR, including glVRC01-class BCRs should increase the chances of activating rare, naïve gl B cells during human immunization.

[0013] An even newer approach in the on-going attempt to elicit bNAbs against HIV through vaccination is based on the ‘germline-targeting’ approach described in the preceding paragraph combined with subsequently guiding the maturation of the first wave of germline antibodies towards their broad neutralizing form along specific evolutionary pathways, using specifically designed ‘booster’ engineered Env. Several attempts have been made to guide the maturation of glBCR stimulated with engineered Env. These strategies include, for example, Tian et al., (2016F053-0192PCT / 23-207-WO-PCT Cell, 166, 1471-1484) who administered: (i) multimerized engineered outer domain (eOD), followed by (ii) multimerized 426c core, followed by (iii) 426c core monomer glycosylated at N463, followed by (iv) 426c core monomer glycosylated at N463 and N460, followed by (v) 426c core monomer glycosylated at N463, N460, and N276 followed by (vi) a soluble wild-type trimeric 426c (i.e., a natural HIV Env); and Briney et al., (2016 Cell 166, 1459-1470) who administered either: (A-i) multimerized eOD, followed by (A-ii) multimerized BG505 GTC core (this protein is similar to the 426c core, but generated from a different strain of HIV) followed by (A-iii) a soluble wild-type trimeric BG505, but for removal of the NLGS site at N276, followed by (A-iv) a repeat administration of the soluble wild-type trimeric BG505, but for removal of the NLGS site at N276 or (B-i) multimerized eOD, followed by (B-ii) a soluble wild-type trimeric BG505, but for removal of NLGS sites N460, N463, and N276 followed by (B-iii) a soluble wild-type trimeric BG505, but for removal of the NLGS site at N276, followed by (B-iv) a repeat administration of the soluble wild-type trimeric BG505, but for removal of the NLGS site at N276. Unfortunately, none of these approaches elicited the maturation of antibodies capable of broadly neutralizing HIV. Tian et al., Cell.166(6):1471-84 e18 (2016); Briney et al., Cell 166, 1459-1470, (2016); Chen et al., Immunity, 54(2), 324-329 (2021).

[0014] Moreover, Tian et al. (2016 Cell, 166, 1471-1484) and Briney et al., (2016 Cell 166, 1459- 1470) employed immunization schedules of at least 20 weeks. Chen et al., (2021 Immunity, 54(2), 324-329) reported neutralization of up to 54% of a 208-strain panel. However, their approach included 9 immunizations over the course of 80 weeks. Reducing the timeline and number of vaccinations for effective immunization would be beneficial to confer immunity more quickly, reduce loss to follow-up, and reduce the spread of HIV during immunization, among other benefits. SUMMARY OF THE DISCLOSURE

[0015] The current disclosure provides immunization schedules that elicit the production ofF053-0192PCT / 23-207-WO-PCT broadly-neutralizing antibodies against the human immunodeficiency virus (HIV), including Tier 2 HIV. The schedules include administration of engineered and / or multimerized antigens, one of which binds VRC01 germline B cell receptor (glBCR) (e.g., 426c core, engineered outer domain (eOD), BG505 SOSIP.v41-GT-1). In particular embodiments, administered antigens are derived from 426c, HxB2, or the consensus group M. The schedule can include administration of engineered and / or multimerized 426c core, HxB2 core, 426c SOSIP, and / or group M consensus SOSIP. In particular embodiments, both the gp120 and gp41 subunits of 426c SOSIP can be derived from 426c (426c wild type SOSIP, 426c WT SOSIP) or the gp120 subunit is derived from 426c and the gp41 subunit from BG505 (chimeric 426c SOSIP, chi 426c SOSIP). In particular embodiments, the schedule includes administration of 3-5 antigens over 9-21 weeks, in protein or nucleic-acid based forms, providing significant advances in efficiency addressing drawbacks described above.

[0016] Particular embodiments include administering the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 wild-type (WT) core, chi-426c SOSIP, and consensus M (ConM) SOSIP.

[0017] Particular embodiments include administering the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 WT core, chi-426c SOSIP, ConM SOSIP, and ConM SOSIP.

[0018] Particular embodiments include administering the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 WT core, and ConM SOSIP.

[0019] Particular embodiments include administering the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 WT core, ConM SOSIP, and ConM SOSIP.

[0020] Particular embodiments include administering the following antigens in the following order: multimerized and engineered 426c core, chi-426c SOSIP, and ConM SOSIP.

[0021] In particular embodiments, there are 4 to 6 weeks between antigen administrations. In particular embodiments, all antigen administrations are completed within a 20-week schedule. In particular embodiments, all antigen administrations are completed within a 14-week schedule. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0022] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0023] FIG. 1. Table of IC50 values for monoclonal antibodies (mAb), G3-1, G3R3-1, G3R3-9,F053-0192PCT / 23-207-WO-PCT GCR3-10, and G3R4-6, isolated after immunization protocols described herein. The IC50 values are the mAb concentration resulting in 50% reduction in infection. MLV was used as a negative control. The neutralizing breadth of each mAb is indicated at the bottom of the table. The immunization protocol for each of the isolated mAbs is as follows: G3-1 from 426c / HxB / 426c Gnti / ConM; G3R3-1 from 426c / HxB / ConM; G3R3-9 from 426c / HxB / ConM; G3R3-10 from 426c / HxB / ConM; and G3R4-6 from 426c / HxB / 426c Gnti.

[0024] FIG.2. Summary of neutralization activity of each of the mAbs), G3-1, G3R3-1, G3R3-9, GCR3-10, and G3R4-6, described in FIG.1, and G3-31.

[0025] FIG.3. Sequences supporting the disclosure. DETAILED DESCRIPTION

[0026] Acquired Immunodeficiency Syndrome (AIDS) is characterized by immunosuppression that results in opportunistic infections and malignancies, wasting syndromes, and central nervous system degeneration. Destruction of CD4+ T-cells, which are critical to immune defense, is a major cause of the progressive immune dysfunction that is the hallmark of AIDS disease progression. The loss of CD4+ T cells seriously impairs the body's ability to fight most pathogens, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.

[0027] AIDS is caused by infection with human immunodeficiency virus (HIV). The HIV genome encodes several structural proteins. The env gene encodes the viral envelope glycoprotein (Env), a 160-kilodalton (kDa) protein. Env is cleaved into an external 120-kDa envelope glycoprotein (gp120) and a transmembrane 41-kDa envelope glycoprotein (gp41). gp120 and gp41 are required for HIV to infect cells.

[0028] Mature gp120 wildtype (wt) proteins have 500 amino acids in the primary sequence. gp120 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kDa. The protein includes five conserved regions (C1-C5) and five regions of high variability (V1-V5). Exemplary sequences of wt gp120 proteins are found in GenBank® (United States Department of Health and Human Services, Bethesda, MD), for example accession numbers AAB05604 and AAD12142. It is understood that there are numerous variations in the sequence of gp120 from what is given in these examples. In particular embodiments, and based on the Hxb2 sequence, V5 includes residues 458-466. One of ordinary skill in the art recognizes, however, that V5 varies among different strains in its precise length, amino acid composition, and glycosylation sites. Reference to residues and mutation positions herein refer to Hxb2 numbering, unless clearly noted to the contrary.F053-0192PCT / 23-207-WO-PCT

[0029] HIV infection begins when gp120 binds to CD4 and other receptors on the surface of a host’s target immune system cells (e.g., CD4+ T-cells, macrophages and dendritic cells). The bound virus then fuses with the target cell and reverse transcribes its RNA genome. The resulting viral DNA integrates into the host cell’s genome and begins to produce new viral RNA, resulting in new viral proteins and virions. The virions leave the originally infected cell to infect new cells. This process kills the originally infected cell.

[0030] Antibodies are proteins that can provide protection against pathogens. Antibodies can bind to a pathogen and are protective when this binding interferes with the normal function of a pathogen. For example, many protective antibodies bind to a portion of a pathogen that blocks the pathogen from entering cells. Antibodies can be attached to the surface of B cells (known as B cell receptors or BCR) but exert most of their protective functions when secreted into the blood.

[0031] HIV-1 neutralizing antibodies are antibodies capable of neutralizing HIV’s infection of host cells. Kumar et al., (ACS Omega 2023, 8, 8, 7252-7261) provides an overview of human HIV-1 neutralizing antibodies against diverse epitopes of HIV-1. HIV antibodies generally target four major areas of the Env protein: (i) the portion of gp41 that is external to the cell, but proximal to the cell membrane; (ii) the CD4 receptor-binding site (CD4-BS) of gp120; (iii) two sites including both carbohydrate and amino acid moieties, one at the base of the “V3” loop and another on the “V1 / V2” loops of the gp120 subunit; and (iv) regions spanning elements of both gp120 and gp41.

[0032] Based on their ontogenies and mode of recognition, CD4-BS broadly neutralizing antibodies (bNAbs) are grouped into two major types: (i) heavy chain complementary determining region three (CDRH3)-dominated; and (ii) variable heavy (VH)-gene-restricted. Antibodies that make contact primarily through their CDRH3 regions are further subdivided into the CH103, HJ16, VRC13 and VRC16 classes while the VH-gene-restricted antibodies include the VRC01- and the 8ANC131-class antibodies.

[0033] Vaccines are designed to increase the immunity of a subject against a particular infection by stimulating B cells to produce antibodies against the targeted infectious agent. Each B cell expresses a unique antibody with unique epitope specificity. The unique antibody expressed by each B cell is generated randomly through genetic recombination. A germline (gl) B cell refers to a B cell that has not yet come in contact with its epitope. gl B cells express a membrane-bound BCR. When the BCR binds its particular epitope, the B cell can rapidly proliferate and mature. During proliferation and maturation, the antibody genes undergo somatic hypermutation, which serves to increase the affinity of epitope binding. The increase in affinity of epitope binding that occurs during B cell maturation is required for effective protection against the pathogen. A single naïve B cell is able to undergo dozens of cell divisions to create thousands of antibody-secretingF053-0192PCT / 23-207-WO-PCT B cells and memory B cells expressing the same antibody, or a related antibody that has been mutated to improve binding to the pathogen. This binding can lead to activation of the B cell and production of protective antibodies.

[0034] For decades, researchers have been trying to develop a vaccine that can induce B cells to produce antibodies that are effective to protect against HIV.

[0035] One of the many important reasons for lack of success is thought to be the inability of the Env proteins used as immunogens to engage gl B cell BCRs that encode, for example, the gl of VRC01-class antibodies (e.g., “immature” or not fully developed antibodies). Indeed, maturation of these antibodies to fully neutralizing antibodies requires that they circumvent steric constraints on Env through extensive somatic hypermutation. For example, HIV-1 has evolved to avoid detection by gl B cells that give rise to VRC01-class bNAbs through development of specific N- linked glycosylation sites (NLGS) (for example, in Loop D and V5 of the gp120 subunit). As a consequence, recombinant Env proteins derived from diverse HIV-1 isolates are ineffective in binding to and stimulating B cells engineered to express the glBCR forms of VRC01-class bNAbs in vitro. Targeted disruption of conserved NLGS at position 276 in Loop D, and at positions 460 and 463 in V5 of the 426c clade C Env, however, permits binding and activation gl B cell lines expressing BCRs of two clonally-related VRC01-class bNAbs, VRC01 and NIH45-46 in vitro. These two BCRs represent a small subset of potential VRC01-class antibody progenitors. Thus, designing immunogens capable of recognizing a larger group of glVRC01-class BCRs should increase the chances of activating rare, naïve glVRC01-class B cells during human immunization. In particular embodiments, loop D includes residues 275-283.

[0036] During the past decades, the generation of novel reagents for the isolation of individual B cells and the establishment of high-throughput methodologies led to the characterization of a plethora of new bNAbs from HIV-1-infected subjects. The structural characterization of such antibodies, combined with information of their ontogenies, have vastly improved the understanding on how such antibodies are generated during natural infection and how they interact with Env. For example, when the VH and VL domains of certain bNAbs (b12, 2G12 and 2F5) are reverted to their predicted, inferred germline forms (from here onward termed ‘germline’ for brevity, unless otherwise noted), those antibodies (Abs) no longer bound the dual-tropic 89.4 Env (Xiao et al., Biochemical and Biophysical Research Communications. 2009;390(3):404-9). This was true for many of the bNAbs that have been isolated since, irrespective of their epitope- specificity and their VH / VL-derivation (Jardine et al., Science.2013;340(6133):711-6; McGuire et al., Journal of Experimental Medicine. 2013;210(4):655-63; Hoot et al., PLoS Pathogens. 2013;9(1):e1003106; Klein et al., Cell. 2013;153(1):126-38; Diskin et al., Science.F053-0192PCT / 23-207-WO-PCT 2011;334(6060):1289-93).

[0037] A hypothesis was put forth that commonly available recombinant Envs are ineffective in eliciting bNAbs because they do not initiate the very first step of that process (Jardine et al., Science. 2013;340(6133):711-6; McGuire et al., Journal of Experimental Medicine. 2013;210(4):655-63). It was also hypothesized that during natural HIV-1 infection, rare viral clones that express Envs with particular features emerge and that they initiate the activation of naïve B cells that express germline BCRs that eventually produce bNAbs. This was confirmed through studies showing that Env clones with particular features can emerge during infection and engage the germline BCRs of bNAbs and activate the corresponding naïve B cells (Liao et al., Nature. 2013;496(7446):469-76; Doria-Rose et al., Nature. 2014;509(7498):55-62). The subsequent maturation of these germline antibodies into their broad neutralizing forms requires the emergence of viruses expressing Env variants of the original ‘germline-binding’ one. In cases where the ‘natural’ Envs that are linked with the development of a particular type of bNAb are known, an immunization scheme can be developed based on those ‘natural’ Envs. In many cases however, such natural Envs are not known. Such is the case of the VRC01-class bNAbs. In those cases, de novo Env immunogens must be designed.

[0038] The 426c core activates B cells expressing the germline BCRs of two VRC01-class antibodies, 3BNC60 and 12A21 (Dosenovic et al., Cell. 2015;161(7):1505-15; McGuire et al., Nature communications. 2016;7:10618), among other glBCR that produce CD4-BS antibodies. The 426c core includes modifications to the clade C 426c Env to allow binding the gl forms of BCR, including VRC01-class antibodies (McGuire et al., Nature communications.2016;7:10618). In particular embodiments, the 426c core is based on the gp120 subunit of the 426c Env; it lacks the variable regions 1, 2 and 3 and lacks three key, conserved NLGS (position N276 in Loop D and positions N460 and N463 in V5). Thus, the 426c core includes elements of both the inner and outer domains of gp120. In that, it differs from the engineered outer domain of gp120 (eOD) construct, which only expresses elements of the outer gp120 domain (Jardine et al., Science. 2013;340(6133):711-6). The structures of 426c core bound to germline 3BNC60 and germline NIH45-46 are reported in Scharf et al., (eLife. 2016;5. doi: 10.7554 / eLife.13783) and a high- resolution structure (2.4 Å) of the germline VRC01 antibody bound to the 426c core was recently obtained.

[0039] A major advance in HIV-1 envelope expression has been the use of stabilized trimers, e.g. by disulfide linkages or other suitable stabilizing mutations. SOSIP trimers are soluble, stabilized, proteolytically cleaved, trimeric proteins that can be generated by engineering an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P,F053-0192PCT / 23-207-WO-PCT within gp41 (IP). SOSIP trimers induce difficult to induce potent (tier 2) autologous HIV-1 neutralizing antibodies.

[0040] The current disclosure provides immunization schedules to successfully guide the maturation of antibodies against human immunodeficiency virus (HIV), including Tier 2 HIV which are the most common circulating strains of HIV and are high priority targets for vaccines. The immunization schedules employ sequential administration of engineered and / or multimerized HIV envelope proteins (Envs) (e.g., antigens) to stimulate germline (gl) and other B cells that can mature to produce bNAbs against HIV. When B cells effectively mature against the HIV virus, subjects will develop bNAbs to mount an effective immune response against HIV infection.

[0041] In particular embodiments, the schedule includes administration of 3-5 antigens over 9-21 weeks, in protein or nucleic-acid based forms, providing significant advances in efficiency and addressing the drawbacks described above.

[0042] In particular embodiments, a first administered antigen binds VRC01 glBCR. This antigen can include an engineered 426c core, an eOD, or BG505 SOSIP.V41-gt-1.

[0043] Particular embodiments administer the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 wild-type (WT) core, 426c SOSIP, and consensus M (ConM) SOSIP.

[0044] Particular embodiments administer the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 WT core, 426c SOSIP, ConM SOSIP, and ConM SOSIP.

[0045] Particular embodiments administer the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 WT core, and ConM SOSIP.

[0046] Particular embodiments administer the following antigens in the following order: multimerized and engineered 426c core, multimerized HXB2 WT core, ConM SOSIP, and ConM SOSIP.

[0047] Particular embodiments administer the following antigens in the following order: multimerized and engineered 426c core, 426c SOSIP, and ConM SOSIP.

[0048] In particular embodiments, there are 4 to 6 weeks between antigen administrations. In particular embodiments, all antigen administrations are complete within a 20-week schedule. In particular embodiments, all antigen administrations are complete within a 14-week schedule.

[0049] In particular embodiments, the 426c SOSIP is a chimeric (chi) 426c SOSIP that includes a gp41 subunit derived from BG505. In particular embodiments, the 426c SOSIP is a wild-type (WT) 426c SOSIP that includes a gp41 subunit derived from 426c.

[0050] Particular embodiments include a four-antigen immunization schedule that includesF053-0192PCT / 23-207-WO-PCT administration of engineered and / or multimerized antigens based on antigens derived from 426c, HxB2, and the consensus group M. The schedule can include administration of an engineered and multimerized 426c core, an HxB2 wild-type core, a chi-426c SOSIP, and a group M consensus SOSIP. In particular embodiments, the schedule includes administration of 4 antigens over the course of 14 weeks.

[0051] Particular embodiments administer an engineered and multimerized 426c core first in the immunization schedule, an HxB2 WT core second in the immunization schedule, a chi-426c SOSIP third in the immunization schedule, and a group M consensus SOSIP fourth in the immunization schedule. Particular embodiments administer an engineered and multimerized 426c core first in the immunization schedule, an HxB2 WT core second in the immunization schedule, and a group M consensus SOSIP third in the immunization schedule. Particular embodiments administer an engineered and multimerized 426c core first in the immunization schedule, a chi- 426c SOSIP second in the immunization schedule, and a group M consensus SOSIP third in the immunization schedule. Particular embodiments administer an engineered and multimerized 426c core first in the immunization schedule, an HxB2 WT core second in the immunization schedule, a first administration of a group M consensus SOSIP third in the immunization schedule, and a second administration of the group M consensus SOSIP fourth in the immunization schedule. Particular embodiments administer an engineered and multimerized 426c core first in the immunization schedule, an HxB2 WT core second in the immunization schedule, a chi-426c SOSIP third in the immunization schedule, and first administration of a group M consensus SOSIP fourth in the immunization schedule, and a second administration of the group M consensus SOSIP fifth in the immunization schedule. In particular embodiments, no other antigens are required or administered to elicit bnAbs against HIV.

[0052] Particular examples of schedules are provided in Tables 1 and 2 below: Table 1. Antigen Administration Schedules. Each row includes a different immunization schedule. Week 0 Week 4 Week 6 Week 10 Week 14 Week 20F053-0192PCT / 23-207-WO-PCT Multimerized Multimerized ConM.SOSIP ConM.SOSIP 426c.Mod. HXB2.WT.Core Core. g p - , p g - administration. Week 0 Week 4 Week 6 Week 10 Week 14 Multimerized Multimerized chi-426c.SOSIP ConM.SOSIP bereplaced with 426c WT SOSIP. All antigens except those designated as GnTI- / - can be administered in protein or nucleic acid (e.g., mRNA) form. GnTI- / - antigens should be administered as protein so that glycosylation can be controlled.

[0054] In particular examples, administration of the disclosed immunization schedules results in broad neutralization of HIV viruses. Table 3 summarizes the neutralization potentials of the fully mature VRC01 monoclonal antibody (mAb) (isolated from an HIV-1-infected person), of the germline version of VRC01, and of antibody G3-1 isolated from knock-in mice immunized according to the immunization schedule: Week 0: C4b multimerized 426c.Mod.Core (protein); Week 4: C4b multimerized HXB2.WT.Core (protein); Week 10: chi426c SOSIP (GnTI- / -) (protein); and Week 14: ConM SOSIP (protein). More particularly, 10-60µg recombinant protein were administered intramuscularly with 50 µl in each hind leg in the quadriceps / biceps of femoris (total volume 100 µl / mouse). Blood was collected every 2 weeks by the retroorbital route into tubes containing 25 µl citrate-phosphate-dextrose solution. Terminal bleeds, draining lymph nodes and spleens were isolated following the last immunization. Envelope-specific B cells were isolated and their VH / VL genes were amplified and sequenced. Monoclonal antibodies were then generatedF053-0192PCT / 23-207-WO-PCT from paired VH / VL genes, and their binding and neutralizing properties were determined.

[0055] The numbers indicate the mAb concentration at which 50% inhibition of infection was recorded (IC50). >50 indicates that neutralization was not recorded at the highest mAb concentration tested of 50 µg / mL. The clades of tier 2 heterologous viruses are shown in parentheses. Neutralization breadth indicates the percentage of viruses neutralized by an antibody out of the total viruses tested. These viruses are representatives of the global phenotypes of circulating viruses (global panel) and can be used to assess the neutralizing potencies and breadths of sera and antibodies. Table 3. Neutralization Potentials. 1 2 3 4 5 6 7 8 9 G3-1 0.91 9.6 4.0 >50 >50 >50 >50 >50 _A3; Column_ _ _ _ 9.10; Column 9: TRO.11.

[0056] IC50 values and a summary of the neutralization activity of antibodies isolated after immunization schedules described herein are represented in FIG.1 and FIG.2, respectively.

[0057] Aspects of the disclosure are now described in more detail as follows: (i) Antibodies and Epitopes; (ii) VRC01 Antibodies; (ii-a) The Heavy Chain (HC) of VRC01-Class Antibodies; (ii-b) The Light Chain (LC) of VRC01-Class Antibodies; (iii) Vaccine Antigens & Protocols; (iv) Multimerization of Antigen; (v) Lipid Nanoparticles; (vi) Compositions; (vii) Vaccine Adjuvants; (viii) Kits; (ix) Methods of Use; (x) Exemplary Embodiments; (xi) Additional Supporting Sequences; and (xii) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0058] (i) Antibodies and Epitopes. Naturally occurring antibody structural units include a tetramer. Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope.F053-0192PCT / 23-207-WO-PCT From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989). Kabat numbering is used herein unless specifically noted otherwise.

[0059] The carboxy-terminal portion of each chain defines a constant region that can be responsible for effector function. Examples of effector functions include: C1q binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), down regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0060] Within full-length light and heavy chains, the variable and constant regions are joined by a "J" region of amino acids, with the heavy chain also including a "D" region of amino acids. See, e.g., Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

[0061] Human light chains are classified as kappa (κ) and lambda (λ) light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including IgM1 and IgM2. IgA is similarly subdivided into subclasses including IgA1 and IgA2.

[0062] Antibodies bind epitopes on antigens. An antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. An epitope includes specific amino acids that contact the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0063] An “epitope” includes any determinant capable of being bound by an antibody. An epitope is a region of a molecule that is bound by an antibody that targets that region of the molecule, and when that region of the molecule is a protein, includes specific residues that directly contact the binding protein. In particular embodiments, an “epitope” denotes the binding site on a protein target bound by a corresponding antibody. The antibody either binds to a linear epitope, (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), or the antibody binds to a three- dimensional structure formed by the spatial arrangement of several short stretches of the protein target. Three-dimensional epitopes recognized by an antibody, e.g., by the epitope recognitionF053-0192PCT / 23-207-WO-PCT site or paratope of an antibody or antibody fragment, can be thought of as three-dimensional surface features of an epitope molecule. These features fit precisely (in)to the corresponding binding site of the antibody and thereby binding between the antibody and its target protein is facilitated. In particular embodiments, an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow an antibody would cast; and (ii) the individual participating side chains and backbone residues. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions.

[0064] (ii) VRC01-Class Antibodies. As indicated previously, based on their ontogenies and mode of recognition, CD4-BS bNAbs are grouped into two major types: CDRH3-dominated (the most common way antibodies bind their epitopes) and VH-gene-restricted (Zhou et al., Cell. 2015;161(6):1280-92). Antibodies that make contact primarily through their CDRH3 regions are further subdivided into the CH103, HJ16, VRC13 and VRC16 classes, while the VH-gene- restricted antibodies, which make contact primarily through their CDRH2 domains, include the VRC01- and the 8ANC131-classes (derived from VH1-2 and VH1-46, respectively).

[0065] At least 29 VRC01-class antibodies have been isolated from at least nine HIV-1+ subjects (Diskin et al., Science. 334(6060):1289-93 (2011); Wu et al., Science. 333(6049):1593-602 (2011); Zhou et al., Immunity.39(2):245-58 (2013); Huang et al., Immunity.45(5):1108-21 (2016); Zhou et al., Cell. 161(6):1280-92 (2015); Kwong & Mascola, Immunity. 37(3):412-25 (2012); Sajadi et al., Cell. 173(7): 1783-1795 (2018); Umotoy et al., Immunity. 51(1): 141-154 (2019); Barnes et al., Sci Adv 8, eabp8155, (2022)).

[0066] (ii-a) The Heavy Chain (HC) of VRC01-Class Antibodies. All known VRC01-class antibodies are derived from one of the five VH1-2 alleles, the VH1-2*02 allele. Three amino acids, Trp50 heavy chain (HC), Asn58HC and Arg71HC, present in the CDRH2 domain of VRC01-class antibodies (i.e., they are encoded by the germline VH1-2 gene segment) make key contacts with Env (Scharf et al., Proceedings of the National Academy of Sciences of the United States of America.2013;110(15):6049-54; West et al., Proceedings of the National Academy of Sciences of the United States of America.2012;109(30):E2083-90; Zhou et al., Immunity.2013;39(2):245- 58). Structural information has revealed the reasons why these three amino acids are critically important for the interaction of the *02 allele with Env: Trp50HC makes contact with the conserved amino acid in Loop D, Asn280; Asn58HC makes contact with the conserved amino acid Arg456 in V5; and Arg71HC makes a key contact with amino acid Asp368 in the CD4-BS. Despite the extensive amino acid changes that occur during affinity maturation of these Abs, these three key HC amino acids remain unaltered (Scharf et al., Proceedings of the National Academy of Sciences of the United States of America. 2013;110(15):6049-54; Zhou et al., Cell.F053-0192PCT / 23-207-WO-PCT 2015;161(6):1280-92; Scharf et al., eLife.2016;5. doi: 10.7554 / eLife.13783). In addition to allele *02, alleles *03 and *04 also express these three amino acids.

[0067] It is also noteworthy that mature VRC01-class Abs have an 11-18 amino acid long CDRH3 and most have a Trp that is located 5 amino acids before the start of FW4 (Trp100BHC on VRC01 numbering). This Trp interacts with Asn279 gp120 via hydrogen-bonding (Scharf et al., Proceedings of the National Academy of Sciences of the United States of America. 2013;110(15):6049-54). This Trp is present in the germline CDRH3 of VRC01-class Abs and are expressed on naïve B cells in HIV-1- subjects (Yacoob et al., Cell Reports.2016;17(6):1560-70).

[0068] (ii-b) The Light Chain (LC) of VRC01-Class Abs. Only a few LC families (Κ3-20, Κ3-15, Κ1-33, K1-5, and λ2-14) are presently known to pair with VH1-2*02 to generate VRC01-class antibodies. Importantly, all the LCs associated with VRC01-class bNAbs express an unusually short (5 amino acid long) CDRL3 region (Zhou et al., Immunity.2013;39(2):245-58; Zhou et al., Cell. 2015;161(6):1280-92; Kwong & Mascola, Immunity. 2012;37(3):412-25). Less than 0.05% of LCs with these properties are present in the human naïve B cell repertoire (Jardine et al., Science.2016;351(6280):1458-63; Sok et al., Science.2016;353(6307):1557-60). The particular angle of approach of VRC01-class Abs requires such a short CDRL3; otherwise, these Abs will not bind Env because of steric clashes with Loop D and V5. Thus, without being bound by theory, the short CDRL3 is presently believed not to be the result of somatic hypermutation but has to exist in the germline form of these antibodies (Zhou et al., Immunity. 2013;39(2):245-58). Accordingly, one of the main goals of ‘germline-targeting’ immunogens is to select for B cells expressing VH1-2*02 VH paired with LCs with 5 amino acid long CDRL3s. Within the 5 amino acid stretch, a key feature of the mature VRC01 Abs is the presence of a negatively charged amino acid, glutamic acid, at position 96. Glu96LC makes key contacts with the V5 loop and Loop D and is one of the amino acids that are linked with the neutralizing activities of VRC01-class Abs. So, ideally, a targeting immunogen should select for LCs with a 5 amino acid long CDRL3 that includes a Glu96. The CDRL1 domains of the mature VRC01-class Abs are also involved in the interaction of these Abs with Env. The mature CDRL1 domains are either shorter (by 2-6 AA) than the corresponding germline domains or contain multiple glycines which provide chain flexibility (Scharf et al., Proceedings of the National Academy of Sciences of the United States of America. 2013;110(15):6049-54; Zhou et al., Immunity.2013;39(2):245-58; Scharf et al., eLife.2016;5. doi: 10.7554 / eLife.13783). The combination of an unusually short CDRL3 (present at the germline level) with a shortening of the CDRL1 (acquired during affinity maturation) allows the mature VRC01-class antibodies to bypass several key steric clashes with Env, in particular carbohydrate moieties that are located in Loop D (conserved position N276). New information from theF053-0192PCT / 23-207-WO-PCT structural analysis of several germline VRC01-class antibodies bound to Env-derived proteins, suggests that the CDRL1 amino acid shortening may not be important for the recognition of Env by glVRC01-class antibodies. In sum, VRC01-class germline antibodies exhibit preformed antigen-binding and conformations and affinity maturation that result in increased induced-fit recognition (Scharf et al., eLife.2016;5. doi: 10.7554 / eLife.13783).

[0069] (iii) Vaccine Antigens & Protocols. In particular embodiments, the present disclosure provides engineered antigens to elicit the production of bNAbs against HIV. An engineered antigen refers to an antigen that is modified or synthetically produced. The engineered antigen can be derived from a wild type antigen or can be computationally designed. According to certain aspects of the current disclosure, at least one administered antigen (e.g., an HIV Env) must be capable of binding and activating glVRC01 B cells.

[0070] In particular embodiments, engineered antigens are derived from antigens of HIV-1 Group M. HIV-1 Group M is a common, widespread group of the HIV virus. Within Group M, there are several subtypes (A, B, C, D, F, G, H, J, and K). Example of Group M HIV include HxB2 and 426c.

[0071] In particular embodiments, an HIV Env that binds and activates glVRC01 B cells includes an HIV Env derived from the wild-type 426c core.426c is a specific HIV-1 Env protein sequence derived from a subtype C strain of HIV-1. A 426c core is a truncated form of the protein, wherein non-essential, variable, and / or flexible regions are removed or minimized. In particular embodiments, a 426c core derived from the wild-type 426c core includes an HIV Env protein with the following mutations, modifications, and characteristics: mutations N460D, N463D, S278R, G471S, V65C, and S115C; no mutation at position 276; removal of V1 and V2; V3 replacement with a flexible linker; an N-terminal truncation before 44; and a C-terminal truncation after 494. In particular embodiments, the mutated 426c core includes the sequence: VWKEAKTTLFCASDAKAYEKECHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQM QEDVISIWDQCLKPCVKLTNTSTLTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNV STVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLRDNAKIIIVQLNKSVEIVCTRPNNGGSGSGG DIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTS GLFNDTISNATIMLPCRIKQIINMWQEVGKAIYAPPIKGNITCKSDITGLLLLRDGGDTTDNTEIFR PSGGDMRDNWRSELYKYKVVEIKPL (SEQ ID NO: 1).

[0072] Particular embodiments of an engineered 426c core (also referred to herein as 426.Mod.Core) include the following mutations: N460D; N463D; S278R; G471S; V65C; S115C; removal of V1 and V2; V3 replacement with a flexible linker; and an N-terminal truncation and a C-terminal truncation. In particular forms of these embodiments, the 426.Mod.Core does not include a mutation at position 276.F053-0192PCT / 23-207-WO-PCT

[0073] Particular embodiments of the 426.Mod.Core include the following mutations: N460D; N463D; S278R; and G471S; removal of V1 and V2; V3 replacement with a flexible linker; and an N-terminal truncation and a C-terminal truncation. In particular forms of these embodiments, the 426.Mod.Core does not include a mutation at position 276. In particular embodiments, V65C and S115C can optionally be included to stabilize the 426.Mod.Core following removal of the V1 and V2 loops.

[0074] Particular embodiments of the 426.Mod.Core include the following mutations: N460D; N463D; S278R; G471S; V65C; S115C; removal of V1 and V2; V3 replacement with a flexible linker; and an N-terminal truncation. In particular embodiments, V1 refers to 131-152 and V2 refers to 161-196. In particular embodiments, removal of V1 and V2 loops includes removal of 123-196. In particular embodiments, V3 refers to 296-331. In particular embodiments, removal of V3 with a flexible linker replacement includes removal of 301-323 and replacement with GGSGSG (SEQ ID NO: 2). In addition to the Gly-Ser linker of SEQ ID NO: 2, a number of flexible linkers can be used. In particular embodiments, a flexible linker is used to replace V3. The linker sequence should not be significantly deleterious to the immunogenicity of the engineered antigens and may even be beneficial to immunogenicity. Such linkers are known to those of skill in the art. One exemplary flexible linker includes Ac-Cys-Gly-Gly-Gly (SEQ ID NO: 3). Additional flexible linkers include GSTSGSGKPGSGEGSTKG (SEQ ID NO: 4) and SGRAHAG (SEQ ID NO: 5). Further examples include a linker that includes (Gly)n, where n=1 to 10 (e.g., n=1, 2, 3, 4 (SEQ ID NO: 6), 5 (SEQ ID NO: 7), 6 (SEQ ID NO: 8), 7 (SEQ ID NO: 9), 8 (SEQ ID NO: 10), 9 (SEQ ID NO: 11), or 10 (SEQ ID NO: 12); (Ser)n, where n=1 to 10 (e.g., n=1, 2, 3, 4 (SEQ ID NO: 13), 5 (SEQ ID NO: 14), 6 (SEQ ID NO: 15), 7 (SEQ ID NO: 16), 8 (SEQ ID NO: 17), 9 (SEQ ID NO: 18), or 10 (SEQ ID NO: 19); (Ala)n, where n=1 to 10 (e.g., n=1, 2, 3, 4 (SEQ ID NO: 20), 5 (SEQ ID NO: 21), 6 (SEQ ID NO: 22), 7 (SEQ ID NO: 23), 8 (SEQ ID NO: 24), 9 (SEQ ID NO: 25), or 10 (SEQ ID NO: 26); (Gly-Ser)n, where n=1, 2 (SEQ ID NO: 27), 3 (SEQ ID NO: 28), 4 (SEQ ID NO: 29), 5 (SEQ ID NO: 30), 6 (SEQ ID NO: 31), 7 (SEQ ID NO: 32), 8 (SEQ ID NO: 33), 9 (SEQ ID NO: 34), or 10 (SEQ ID NO: 35); (Gly-Ser-Ser-Gly)n, where n=1 (SEQ ID NO: 36), 2 (SEQ ID NO: 37), 3 (SEQ ID NO: 38), 4 (SEQ ID NO: 39), 5 (SEQ ID NO: 40), 6 (SEQ ID NO: 41), 7 (SEQ ID NO: 42), 8 (SEQ ID NO: 43), 9 (SEQ ID NO: 44), or 10 (SEQ ID NO: 45); (Gly-Ser-Gly)n, where n=1, 2 (SEQ ID NO:46), 3 (SEQ ID NO: 47), 4 (SEQ ID NO: 48), 5 (SEQ ID NO: 49), 6 (SEQ ID NO: 50), 7 (SEQ ID NO: 51), 8 (SEQ ID NO: 52), 9 (SEQ ID NO: 53), or 10 (SEQ ID NO: 54); (Gly-Ser-Ser)n, where n=1, 2 (SEQ ID NO: 55), 3 (SEQ ID NO: 56), 4 (SEQ ID NO: 57), 5 (SEQ ID NO: 58), 6 (SEQ ID NO: 59), 7 (SEQ ID NO: 60), 8 (SEQ ID NO: 61), 9 (SEQ ID NO: 62), or 10 (SEQ ID NO: 63); (Gly-Ala)n, where n=1, 2 (SEQ ID NO: 64), 3 (SEQ ID NO: 65), 4 (SEQ IDF053-0192PCT / 23-207-WO-PCT NO: 66), 5 (SEQ ID NO: 67), 6 (SEQ ID NO: 68), 7 (SEQ ID NO: 69), 8 (SEQ ID NO: 70), 9 (SEQ ID NO: 71), or 10 (SEQ ID NO: 72); or any combination thereof.

[0075] Particular embodiments exclude a mutation at position 276. In the presence of the S278R mutation, the unmutated 276 position is not glycosylated.

[0076] An N-terminal truncation refers to a truncation at the N-terminal end of a naturally- occurring Env. In particular embodiments, the N-terminal truncation is before residue 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 or 39. In particular embodiments, the N-terminal truncation is before residue 46, 45, 44, 43 or 42. In particular embodiments, the N-terminal truncation is before residue 44.

[0077] Particular embodiments include a C-terminal truncation. In particular embodiments, the C- terminal truncation is after residue 499, 498, 497, 496, 495, 494, 493, 492, 491, 490 or 389. In particular embodiments, the C-terminal truncation is after residue 496, 495, 494, 493 or 492. In particular embodiments, the C-terminal truncation is after residue 494.

[0078] In particular embodiments, a key mutation on the 426.Mod.Core that is required for glVRC01 binding knocks-out N276. In particular embodiments, the NLGS at position N460 should be eliminated. In particular embodiments, the N463 on the 426.Mod.Core may be retained. In particular embodiments, the glycans at N463 on the 426.Mod.Core can stabilize the binding of glVRC01 to the 426.Mod.Core that lacks N276. One reason why glVRC01 binds the 426.Mod.Core once N276 is knocked out is because the 426c Env naturally lacks a conserved NLGS at position 234. It is possible that N234, when glycosylated, may block the binding of glVRC01 even when N276 is knocked out.

[0079] Antigens derived from the 426c core are preferred first antigens within the immunization schedules disclosed herein. In particular embodiments, however, use of an engineered 426c core as the first antigen can be replaced with a different antigen capable of binding VRC01 glBCR. These alternative antigen include eOD described in WO2016 / 205704 and the BG505 SOSIP.v4.1-GT1 trimer described in Medina-Ramierz et al., J Exp Med 214(9), 2573-2590 (2017).

[0080] In particular embodiments, an engineered antigen includes a SOSIP antigen. In particular embodiments, the 426c SOSIP includes the following mutations, modifications, and characteristics: an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (IP) (426c wild-type (WT) SOSIP). Particular embodiments of the 426c SOSIP lack the N276, N460 and N463 NLGS (426c DS-SOSIP D3). In particular embodiments, the 426c SOSIP lacks the N276 glycan (426c Δ276 DS-SOSIP). In particular embodiments, a 426c SOSIP is a chimeric (chi) 426c SOSIP that includes a gp41 subunit derived from BG505. In particular embodiments, the chi-426c SOSIP includes theF053-0192PCT / 23-207-WO-PCT sequence: AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTEN FNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLG EIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTCTQACPKVTFDPIP IHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLSD NAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKK KLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMW QEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTTNNTEIFRPGGGDMRDNWRSELYKYKVVKIE PLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQ SNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWS NRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGSGGSG (SEQ ID NO: 164).

[0081] In particular embodiments, the 426c wild-type SOSIP includes the sequence: GNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENF NMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEI KNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTLTQACPKVTFDPIPIH YCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLSDNA KIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLK EHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEV GKAIYAPPIKGNITCKSDITGLLLLRDGGNTTNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLG VAPTDCKRRVVERRRRRRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLL RAPEAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICCTAVPWNISWSNKSK EEIWENMTWMQWDREINNYTNTIYRLLEESQNQQENNEKDLLALD (SEQ ID NO: 172).

[0082] In particular embodiments, an antigen of the immunization schedules disclosed herein includes the wild-type HXB2 core. HXB2 is a reference strain of HIV-1 Group M, subtype B. The HXB2 core refers to a truncated form of the HIV-1 gp120 protein from HXB2. The wild-type HXB2 core may be referred to as Hxb2core-gp120. In particular embodiments, the HXB2 core includes the sequence: VWKEATTTLFCASDAKAYDTECHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQM HEDIISLWDQCLKPCVKLTNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTRPNNGGSGSGG NMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNST QLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLL LTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPL (SEQ ID NO: 161).F053-0192PCT / 23-207-WO-PCT

[0083] In particular embodiments, an antigen of the immunization schedules disclosed herein includes a consensus M sequence (ConM). The ConM sequence represents a computational derived sequence that represents the most common (or consensus) amino acid or nucleotide at each position in a HIV-1 gene or fragment thereof of HIV-1 Group M. In particular embodiments, ConM includes an HIV Env protein with the following mutations, modifications, and characteristics: an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (IP) (ConM SOSIP). In particular embodiments, the ConM SOSIP includes the sequence: AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEKRNVWATHCCVPTDPNPQEIVLENVTEN FNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLNCTDVNATNNTTNNEEIKNCSFNITTE LRDKKKKVYALFYKLDVVPIDDNNSYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCNDK KFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENITNNAKTIIVQLNESVEINCTR PNNNTRKSIRIGPGQWFYATGDIIGDIRQAHCNISRTKWNKTLQQVAKKLREHFNKTIIFNPSSG GDLEITTHSFNCGGEFFYCNTSELFNSTWNGTNNTITLPCRIKQIINMWQRVGQAMYAPPIEGKI RCTSNITGLLLTRDGGNNNTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVE RRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQHLLQL TVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICCTNVPWNSSWSNKSQDEIWDNMTWM EWDKEINNYTDIIYSLIEESQNQQEKNEQELLALDGGSGGSG (SEQ ID NO: 168).

[0084] In particular embodiments, antigen can be delivered as a recombinant protein or as a nucleic acid encoding a recombinant protein. In particular embodiments, an immunization schedule includes more than one antigen delivered as a recombinant protein. In particular embodiments, an immunization schedule includes more than one antigen delivered as nucleic acids. In particular embodiments, an immunization schedule includes antigen delivery as a recombinant protein and as nucleic acids. For example, an immunization schedule may include a first antigen delivered as a nucleic acid and a second antigen delivered as a recombinant protein.

[0085] Nucleic acids and their equivalents refer to molecules that include a nitrogenous base, a sugar, and a phosphate group. A nucleotide is a monomer of DNA or RNA. A nucleotide, for instance, is a chemical structure. Nucleic acids refer to linked monomers of DNA or RNA.

[0086] DNA refers to a polymer of nucleotides (also referred to as “nucleobases”) including deoxyribose. The nucleotides in DNA include cytosine (C), guanine (G), adenine (A), and thymine (T). Each DNA nucleotide includes a deoxyribose and a phosphate group. An example single- stranded DNA (ssDNA) molecule includes a chain of covalently bonded DNA nucleotides. In the example ssDNA molecule, the phosphate group of the mth nucleotide is covalently bonded to the deoxyribose of the (m-1)th nucleotide, wherein m is a positive integer greater than 2 and less thanF053-0192PCT / 23-207-WO-PCT or equal to the number of DNA nucleotides in the chain. In various examples, DNA is double- stranded and includes two ssDNA molecules that are complementary to one another and coiled around each other in a double helix form. The nucleotides of one ssDNA molecule are hydrogen bonded to the nucleotides of the other ssDNA molecule. In particular, the pyrimidines (A and T) hydrogen bond to each other, and the purines (C and G) hydrogen bond to each other.

[0087] RNA refers to a polymer of nucleotides containing ribose. The nucleotides in RNA include cytosine (C), guanine (G), adenine (A), and uracil (U). Each RNA nucleotide includes a ribose and a phosphate group. In an example RNA molecule, the phosphate group of the nth nucleotide is covalently bonded to the ribose of the (n-1)th nucleotide, wherein n is a positive integer greater than 2 and less than or equal to the number of RNA nucleotides in the chain. Messenger RNA (mRNA) is a type of RNA molecule that is synthesized (or “transcribed”) by RNA polymerase to be complementary to a gene encoded in a DNA sequence and is also used by a ribosome to synthesize a polypeptide or protein. An mRNA is therefore an example of a “coding RNA.” MicroRNA (“miRNA”) are single-stranded RNA molecules that perform post-transcriptional gene expression regulation. For instance, a miRNA may bind to a complementary mRNA molecule, thereby cleaving, destabilizing, or otherwise preventing the mRNA molecule from being translated into a polypeptide or protein by a ribosome. In various examples, a miRNA has a length in a range of 21 to 23 RNA nucleotides. As used herein, the terms “non-coding RNA” may refer to a type of RNA that is not translated into a protein. Examples of non-coding RNA include miRNA, transfer RNA (tRNA), and ribosomal RNA (rRNA). The term “functional RNA,” and its equivalents, may refer to any RNA molecule that impacts a biological process. For instance, functional RNA may include mRNA, miRNA, tRNA, and rRNA.

[0088] Particular embodiments utilize as a nucleic acid self-amplifying RNA (saRNA), in vitro transcribed RNA, circular RNA, plasmid DNA, minicircle DNA, or dogpybone DNA (dbDNA).

[0089] “Self-amplifying RNA” (also referred to as “self-replicating RNA”) refers to an RNA molecule that can amplify itself in a host cell, leading to an amplification of the amount of RNA encoding a desired gene product and enhanced efficiency of expression of the encoded gene product (e.g., antigen). In particular embodiments, self-amplifying RNA refers to self-amplifying mRNA and / or replicon RNA. In particular embodiments self-amplifying RNA includes nucleotide sequences encoding all components necessary for replication, e.g., a replicase and / or transcriptase. The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex, thus creating genomic negative-strand copies of positive-strand RNA.

[0090] In particular embodiments, self-amplifying RNA derived from a viral genome that includes viral genes encoding non-structural proteins important for viral replication and that lacks viralF053-0192PCT / 23-207-WO-PCT genes encoding structural proteins. A self-replicating RNA can encode further subgenomic RNAs that are not able to self-replicate. Self-amplifying RNAs are found in positive-strand RNA viruses and use their own encoded viral polymerase to amplify itself, e.g., as described in Johanning et al., Nucleic Acids Res., 23(9):1495-1501 (1995); Khromykh et al., Current Opinion in Molecular Therapeutics, 2(5):556-570 (2000); and Smerdou et al., Current Opinion in Molecular Therapeutics, 1(2):244-251 (1999).

[0091] In particular embodiments, self-amplifying RNA may be encoded by DNA. Such a DNA molecule can be placed under control of a constitutive or inducible promoter. Activation of the activatable promoter leads to the production of the self-amplifying RNA.

[0092] In particular embodiments of the disclosure, nucleic acids are delivered to cells through the use of a lipid nanoparticle, described elsewhere herein.

[0093] In particular embodiments the HxB2 wild-type core is administered between 21 and 35 days after administration of the engineered and multimerized 426c core. In particular embodiments the HxB2 wild-type core is administered between 24 and 31 days after administration of the engineered and multimerized 426c core. In particular embodiments the HxB2 wild-type core is administered between 26 and 29 days after administration of the engineered and multimerized 426c core. In particular embodiments the HxB2 wild-type core is administered 28 days after administration of the engineered and multimerized 426c core.

[0094] In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 35 and 77 days after administration of the engineered and multimerized 426c core. In particular embodiments the 426c SOSIP (e.g., ch-426c SOSIP) is administered between 35 and 49 days after administration of the engineered and multimerized 426c core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 38 and 45 days after administration of the engineered and multimerized 426c core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered 42 days after administration of the engineered and multimerized 426c core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 63 and 77 days after administration of the engineered and multimerized 426c core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 66 and 73 days after administration of the engineered and multimerized 426c core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered 70 days after administration of the engineered and multimerized 426c core.

[0095] In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 35 and 49 days after administration of the HxB2 wild-type core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 32 and 45 daysF053-0192PCT / 23-207-WO-PCT after administration of the HxB2 wild-type core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 29 and 43 days after administration of the HxB2 wild- type core. In particular embodiments the 426c SOSIP (e.g., chi-426c SOSIP) is administered between 42 days after administration of the HxB2 wild-type core.

[0096] In particular embodiments the group M consensus SOSIP is administered between 63 and 147 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered between 63 and 77 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered between 66 and 73 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered 70 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered between 91 and 105 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered between 94 and 101 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered 98 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered between 133 and 147 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered between 136 and 143 days after administration of the engineered and multimerized 426c core. In particular embodiments the group M consensus SOSIP is administered 140 days after administration of the engineered and multimerized 426c core.

[0097] In particular embodiments the group M consensus SOSIP is administered between 35 and 119 days after administration of the HxB2 wild-type core. In particular embodiments the group M consensus SOSIP is administered between 35 and 49 days after administration of the HxB2 wild- type core. In particular embodiments the group M consensus SOSIP is administered between 38 and 45 days after administration of the HxB2 wild-type core. In particular embodiments the group M consensus SOSIP is administered 42 days after administration of the HxB2 wild-type core. In particular embodiments the group M consensus SOSIP is administered between 63 and 77 days after administration of the HxB2 wild-type core. In particular embodiments the group M consensus SOSIP is administered between 66 and 73 days after administration of the HxB2 wild-type core. In particular embodiments the group M consensus SOSIP is administered 70 days after administration of the HxB2 wild-type core. In particular embodiments the group M consensus SOSIP is administered between 105 and 119 days after administration of the HxB2 wild-type core.F053-0192PCT / 23-207-WO-PCT In particular embodiments the group M consensus SOSIP is administered between 108 and 115 days after administration of the HxB2 wild-type core. In particular embodiments the group M consensus SOSIP is administered 112 days after administration of the HxB2 wild-type core.

[0098] In particular embodiments the group M consensus SOSIP is administered between 21 and 77 days after administration of the 426c SOSIP (e.g., chi-426c SOSIP). In particular embodiments the group M consensus SOSIP is administered between 21 and 35 days after administration of the 426c SOSIP (e.g., chi-426c SOSIP). In particular embodiments the group M consensus SOSIP is administered between 24 and 31 days after administration of the 426c SOSIP (e.g., chi- 426c SOSIP). In particular embodiments the group M consensus SOSIP is administered 24 days after administration of the 426c SOSIP (e.g., chi-426c SOSIP). In particular embodiments the group M consensus SOSIP is administered between 63 and 77 days after administration of the 426c SOSIP (e.g., chi-426c SOSIP). In particular embodiments the group M consensus SOSIP is administered between 66 and 73 days after administration of the 426c SOSIP (e.g., chi-426c SOSIP). In particular embodiments the group M consensus SOSIP is administered 70 days after administration of the 426c SOSIP (e.g., chi-426c SOSIP).

[0099] Particular embodiments of immunization schedules disclosed herein include administration of less than 9 antigens. Particular embodiments of immunization schedules disclosed herein include administration of 8 antigens. Particular embodiments of immunization schedules disclosed herein include administration of 7 antigens. Particular embodiments of immunization schedules disclosed herein include administration of 6 antigens. Particular embodiments of immunization schedules disclosed herein include administration of 5 antigens. Particular embodiments of immunization schedules disclosed herein include administration of 4 antigens. Particular embodiments of immunization schedules disclosed herein include administration of 3 antigens.

[0100] Particular embodiments of immunization schedules disclosed herein include administration of less than 7 antigens in less than 40 weeks, less than 30 weeks, or less than 25 weeks. Particular embodiments of immunization schedules disclosed herein include administration of 5 antigens within 21 weeks. Particular embodiments of immunization schedules disclosed herein include administration of 5 antigens within 20 weeks. Particular embodiments of immunization schedules disclosed herein include administration of 5 antigens within 16 weeks. Particular embodiments of immunization schedules disclosed herein include administration of 4 antigens within 14 weeks.

[0101] (iv) Multimerization of Antigens. In particular embodiments, antigens disclosed herein can be multimerized. A multimerized antigen refers to an assembly of two or more antigens or relevantF053-0192PCT / 23-207-WO-PCT portions thereof. Multimerization can enhance the immunogenicity of an administered antigen.

[0102] In particular embodiments, antigens can be multimerized with a C4b multimerization domain. C4 binding protein (C4b) is the major inhibitor of the classical complement and lectin pathway. The complement system is a major part of innate immunity and is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune responses when necessary. It also recognizes necrotic or apoptotic cells (Hofmeyer et al., Journal of Molecular Biology.2013 Apr 26;425(8):1302-17).

[0103] Full-length native C4b includes seven α-chains linked together by a multimerization (i.e., heptamerization) domain at the C-terminus of the α-chains (Blom et al., (2004) Molecular Immunology 40: 1333–1346). One of the α-chains can be replaced by a β-chain in humans. The wild-type C4b multimerization domain is 57 amino acid residues in humans and 54 amino acid residues in mice (Forbes et al., PloS One.2012; 7(9): e44943). It contains an amphipathic α-helix region, which is necessary and sufficient for heptamerization, as well as two cysteine residues which stabilize the structure (Kask et al., (2002) Biochemistry 41: 9349–9357).

[0104] The sequences of a number of C4b domain proteins are available in the art. These include human C4b multimerization domains as well as a number of homologues of human C4b multimerization domains available in the art. There are two types of homologues: orthologues and paralogues. Orthologues are defined as homologous genes in different organisms, i.e. the genes share a common ancestor coincident with the speciation event that generated them. Paralogues are defined as homologous genes in the same organism derived from a gene, chromosome or genome duplication, i.e. the common ancestor of the genes occurred since the last speciation event.

[0105] GenBank® (United States Department of Health and Human Services) indicates mammalian C4b multimerization domain homologues in species including chimpanzees, rhesus monkeys, rabbits, rats, dogs, horses, mice, guinea pigs, pigs, chicken, and cattle. Further C4b multimerization domains may be identified by searching databases of DNA or protein sequences, using commonly available search programs such as BLAST® (National Library of Medicine, Bethesda, MD).

[0106] In particular embodiments, a C4b multimerization domain includes a sequence as set forth in any one of SEQ ID NOs: 122-154. SEQ ID Sequence SF053-0192PCT / 23-207-WO-PCT 123 KKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKLKVELQGLSKE 124 ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKE L E K V K E S H S E E ondomain which includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucines at positionsF053-0192PCT / 23-207-WO-PCT 29, 34, 36, and / or 41; (iv) tyrosine at position 32; (v) lysine at position 33; and / or (vi) cysteines at positions 6 and 18. In particular embodiments, the C4b multimerization domain will be a multimerization domain which includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucines at positions 29, 34, 36, and 41; (iv) tyrosine at position 32; (v) lysine at position 33; and (vi) cysteines at positions 6 and 18.

[0108] C4b multimerization domains can include any of SEQ ID NOs: 122-154 with an N-terminal deletion of at least 1 consecutive amino acid residue(s) (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length. Additional embodiments can include a C-terminal deletion of at least 1 consecutive amino acid residue(s) (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length.

[0109] Particular C4b multimerization domain embodiments will retain or will be modified to include at least 1 of the following residues: A6, E11, A13, D21, C22, P25, A27, E28, L29, R30, T31, L32, L33, E34, I35, K37, L38, L40, E41, I42, Q43, K44, L45, E48, L49, or Q50. Further embodiments will retain or will be modified to include A6, E11, A13, D21, C22, P25, A27, E28, L29, R30, T31, L32, L33, E34, I35, K37, L38, L40, E41, I42, Q43, K44, L45, E48, L49, and Q50. Particular C4b multimerization domain embodiments will include the amino acid sequence: AELR (SEQ ID NO: 155).

[0110] Particular embodiments can utilize a heptamerization domain such as: SEQ Sequence ID NO: Qion domains include: SEQ Se uence K K I K E Y EIF053-0192PCT / 23-207-WO-PCT VWKEAKTTLFCASDAKAYEKECHNVWATHACVPTDPNPQEVVLENVTENFNMWK 160 NDMVDQMQEDVISIWDQCLKPCVKLTNTSTLTQACPKVTFDPIPIHYCAPAGYAILK CNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLRDNAKIII K E Y V 3D,, , , , e o a o a , epace e a e e e , an N-terminal truncation before 44, a C-terminal truncation after 494, and a C4b multimerization domain. This engineered 426c.mod.core excludes a mutation at position 276, but nonetheless lacks N276 glycosylation due to the S278R mutation.

[0113] Particular embodiments can utilize ferritin as a multimerization domain. Ferritin is an iron storage protein found in almost all living organisms and has been extensively studied and engineered for a number of biochemical / biomedical purposes (US 20090233377; Meldrum, et al. Science 257, 522-523 (1992); U.S.20110038025; Yamashita, Biochim Biophys Acta 1800, 846- 857 (2010), including as a multimerizing vaccine platform for displaying peptide epitopes (US 20060251679 (2006); Li, et al. Industrial Biotechnology 2, 143-147 (2006)). Ferritin is particularly useful for multimerizing vaccine epitopes because of its self-assembly and multivalent presentation of the epitopes which induces stronger B cell responses than monovalent forms and induces T-cell independent antibody responses (Bachmann et al., Annual Review of Immunology 15, 235-270 (1997); Dintzis et al. Proceedings of the National Academy of Sciences of the United States of America 73, 3671-3675 (1976)). Furthermore, the molecular architecture of ferritin, which can include 24 subunits assembling into an octahedral cage with 432 symmetry, has the ability to display multimeric antigens on its surface.

[0114] Particular embodiments utilize a monomeric ferritin subunit protein linked to an antigen. The monomeric ferritin subunit protein can include a domain that allows the fusion protein to self- assemble into particles. The monomeric ferritin subunit protein can be selected from a bacterial ferritin, a plant ferritin, an algal ferritin, an insect ferritin, a fungal ferritin, and a mammalian ferritin. In particular embodiments, the monomeric ferritin can be a monomeric subunit of a Helicobacter pylori ferritin protein.

[0115] In particular embodiments, ferritin proteins from different sources (e.g., species) can be fused to form hybrid ferritins or ferritin fusion sequences. An exemplary hybrid includes the Helicobacter pylori-bullfrog ferritin fusion protein described in Kanekiyo et al., (Cell.2015 Aug 27; 162(5): 1090–1100). In particular embodiments, ferritin can include any one of SEQ ID NOs: 117-F053-0192PCT / 23-207-WO-PCT 120. SEQ ID Sequence NO: 117 ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAA D K N N F N :SEQ ID Sequence I A F M E G pidnanoparticle for administration. The term “lipid nanoparticle”, also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and which includes one or more lipids.

[0117] Exemplary LNP include liposomes (microscopic vesicles including at least one concentric lipid bilayer surrounding an aqueous core), liposomal nanoparticles (a liposome structure used to encapsulate another smaller nanoparticle within its core); and lipid-like nanoparticles (liposome- like structures that lack the continuous lipid bilayer characteristic of liposomes).

[0118] An LNP may include any lipid capable of forming a particle to which one or more antigen (e.g., nucleic acid molecules) can be attached or encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generallyF053-0192PCT / 23-207-WO-PCT characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0119] In particular embodiments, the LNP includes one or more cationic lipids. The cationic lipid can be cationisable, i.e., it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. When positively charged, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid includes a zwitterionic lipid that assumes a positive charge on pH decrease. The LNP may include any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0120] In certain embodiments, the LNP may include any further cationic or cationisable lipid, i.e., any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N—(N′,N′dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1- (2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE).

[0121] Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes including DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes including N-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N- dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids including dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA).

[0122] In particular embodiments, the further cationic lipid is an amino lipid. Suitable amino lipidsF053-0192PCT / 23-207-WO-PCT include those described in WO2012 / 016184. Representative amino lipids include 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy- 3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy- 3dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta- 6,9,28,31-tetraen-19-yl 4-(dimethyl amino)butanoate (DLin-MC3-DMA), and 2,2-dilinoleyl-4-(2- dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA).

[0123] Particular embodiments utilize an amino lipid selected from: 2-(Di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol, Dinonyl 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate, Nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoateF053-0192PCT / 23-207-WO-PCT, Heptadecan-9-yl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, Di(heptadecan-9-yl) 8,8'-((2-hydroxyethyl)azanediyl)dioctanoateF053-0192PCT / 23-207-WO-PCT, Heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate,F053-0192PCT / 23-207-WO-PCT Heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4-oxobutyl)amino)octanoate, Henicosan-11-yl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate, or8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoic acid.,

[0124] In particular embodiments, the cationic lipid has a Formula (I), (II) or (III):F053-0192PCT / 23-207-WO-PCTthereof, wherein R1a, R1b, R2a, are as defined herein;or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8, R9, L1, L2, G1, G2, G3, a, b, c and d are as defined herein;F053-0192PCT / 23-207-WO-PCT or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein R1, R2, R3, L1, L2, G1, G2, and G3are as defined herein.

[0125] In particular embodiments, the LNP includes one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0126] In some embodiments, LNP include a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids.

[0127] In some embodiments LNP include a phospholipid. In some embodiments, the phospholipid includes 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn- glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.

[0128] In some embodiments, the phospholipid includes 1-myristoyl-2-palmitoyl-sn-glycero-3- phosphocholine (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC), 1- palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC, PMPC), 1-palmitoyl-2- stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC, PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (16:0-18:1 PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (14:0-22:6 PC), 1-stearoyl-2- myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycero- 3-phosphocholine (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0- 18:1 PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC), 1-stearoyl- 2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4 PC), 1-stearoyl-2-docosahexaenoyl-sn- glycero-3-phosphocholine (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholineF053-0192PCT / 23-207-WO-PCT (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0 PC, OSPC), 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE, POPE), 1-palmitoyl-2-linoleoyl-sn- glycero-3-phosphoethanolamine (16:0-18:2 PE), 1-palmitoyl-2-arachidonoyl-sn-glycero-3- phosphoethanolamine (16:0-20:4 PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3- phosphoethanolamine (16:0-22:6 PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1 PE), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2 PE), 1- stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (18:0-20:4 PE), 1-stearoyl-2- docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0-22:6 PE), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and any combination thereof.

[0129] In some embodiments, the LNP include a structural lipid. In some embodiments, the structural lipid is cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or mixtures thereof.

[0130] In some embodiments, the LNP include a PEG lipid. In some embodiments, the PEG lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, or mixtures thereof.

[0131] In some embodiments, the LNP include an ionizable lipid selected from 3- (didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2- (didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25- ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin- K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethyl amino)butanoate (Dlin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (Dlin-KC2-DMA), 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1- amine (Octyl-CLinDMA (2R)), and (2 S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl- 3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)).

[0132] In some embodiments, the LNP include a quaternary amine compound. In some embodiments, the quaternary amine compound includes 1,2-dioleoyl-3-trimethylammonium- propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-F053-0192PCT / 23-207-WO-PCT N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N-(1,2-dioleoyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DOME), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLePC), 1,2-distearoyl-3- trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP), 1,2-dimyristoyl-3-trimethylammonium- propane (DMTAP), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSePC), 1,2-dipalmitoyl- sn-glycero-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOePC), 1,2-di-(9Z-tetradecenoyl)- sn-glycero-3-ethylphosphocholine (14:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.

[0133] In particular embodiments, LNP include liposomes and lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. As indicated, in particular embodiments, liposomes provide a lipid composition that is an outer layer surrounding a particle.

[0134] Liposomes can be neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other type of bipolar lipids including dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol and (DOPG),F053-0192PCT / 23-207-WO-PCT dicetylphosphate that are able to form vesicles. In particular embodiments, lipids used to create liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE.

[0135] Methods of forming liposomes are described in, for example, US Patent Nos.4,229,360; 4,224,179; 4,241,046; 4,737,323; 4,078,052; 4,235,871; 4,501,728; and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng.9:467 (1980) and Hope et al., Chem. Phys. Lip.40:89 (1986).

[0136] In the context of the present disclosure, LNP are not restricted to any particular morphology, and should be interpreted to include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and / or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of an LNP.

[0137] The size of LNP can vary over a wide range and can be measured in different ways. For example, LNP can have a minimum dimension of 100 nm. The LNP of the present disclosure can also have a minimum dimension of equal to or less than 1000 nm, less than 500 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In particular embodiments, the LNP can have a minimum dimension ranging between 5 nm and 1000 nm, between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm. In particular embodiments, the dimension is the diameter of the LNP. In particular embodiments, a population of LNP can have a mean minimum dimension of equal to or less than 1000 nm, less than 500 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In particular embodiments, a population of LNP in a foam can have a mean diameter ranging between 5 nm and 1000 nm, between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm.

[0138] In various embodiments, the LNP have a mean diameter of from 30 to 1000 nm, 30 nm to 150 nm, from 40 nm to 150 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from 70 nm to 110 nm, from 70 nm to 100 nm, from 80 nm to 100 nm, from 90 nm to 100 nm, from 70 to 90 nm, from 80 nm to 90 nm, from 70 nm to 80 nm, or 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certainF053-0192PCT / 23-207-WO-PCT embodiments, the nucleic acid, when present in the LNP, is resistant in aqueous solution to degradation with a nuclease. Dimensions of the LNP can be determined using, e.g., conventional techniques, such as dynamic light scattering and / or electron microscopy. In particular embodiments, the mean diameter may be represented by the z-average as determined by dynamic light scattering.

[0139] In some embodiments, the nucleic acid or a portion thereof, is encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or all of the lipid portion of the LNP, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response. In some embodiments, the nucleic acid or a portion thereof is associated with the LNP.

[0140] In particular embodiments lipid nanoparticles can be used to deliver self-amplifying RNA, plasmid DNA, minicircle DNA, in vitro transcribed RNA, circular RNA, dogpybone DNA (dbDNA), siRNA, or guide RNA.

[0141] (vi) Compositions. Antigens can be provided as part of compositions formulated for administration to subjects. The formulated antigens can include a recombinant protein and / or a nucleic acid, either or both optionally provided within a nanoparticle. Regardless of particular form, antigens can be referred to as active ingredient(s).

[0142] In particular embodiments, active ingredients are provided as part of a composition that can include, for example, at least 0.1% w / v or w / w of active ingredient(s), at least 1% w / v or w / w of active ingredient(s), at least 10% w / v or w / w of active ingredient(s), at least 20% w / v or w / w of active ingredient(s), at least 30% w / v or w / w of active ingredient(s), at least 40% w / v or w / w of active ingredient(s), at least 50% w / v or w / w of active ingredient(s), at least 60% w / v or w / w of active ingredient(s), at least 70% w / v or w / w of active ingredient(s), at least 80% w / v or w / w of active ingredient(s), at least 90% w / v or w / w of active ingredient(s), at least 95% w / v or w / w of active ingredient(s), or at least 99% w / v or w / w of active ingredient(s).

[0143] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage or ingestion. The compositions can further be formulated for, for example, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and / or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and / or subcutaneous injection.

[0144] For injection, compositions can be formulated as aqueous solutions, such as in buffersF053-0192PCT / 23-207-WO-PCT including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the formulation can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0145] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g. lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0146] For administration by inhalation, compositions can be formulated as aerosol sprays from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic and a suitable powder base such as lactose or starch.

[0147] Any composition formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic or other untoward reactions that outweigh the benefit of administration, whether for research or immunization efforts. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by United States FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0148] Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants,F053-0192PCT / 23-207-WO-PCT antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and / or lubricants.

[0149] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and / or trimethylamine salts.

[0150] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

[0151] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol or mannitol.

[0152] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers or polysaccharides.

[0153] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0154] As indicated, in particular embodiments, an antigen may be encapsulated in or conjugated to nanoparticles for administration. Nanoparticles can include lipid nanoparticles, a polymeric nanoparticle, surfactant-based emulsions, micelles, silica nanoparticles, albumin nanoparticles, nanotubes (e.g., carbon nanotubes), dendrimers, microparticles , and / or nanoparticles that are developed using a combination of nanomaterials such as lipid-polymer nanoparticles.

[0155] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers containing at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release active ingredients following administration for a few weeks up to over 100 days.

[0156] (vii) Vaccine Adjuvants. Particular embodiments include administering a vaccine adjuvant with one or more antigen administrations of an immunization schedule disclosed herein. The term “vaccine adjuvant” refers to material that enhances the immune response to an antigen and is used herein in the customary use of the term. The precise mode of action is not understood for all adjuvants, but such lack of understanding does not prevent their clinical use for a wide varietyF053-0192PCT / 23-207-WO-PCT of vaccines.

[0157] Exemplary vaccine adjuvants include any kind of Toll-like receptor ligand or combinations thereof (e.g. CpG, Cpg-28 (a TLR9 agonist), Polyriboinosinic polyribocytidylic acid (Poly(I:C)), Adjuplex (a biodegradable matrix of carbomer homopolymer-Carbopol-and nanoliposomes), α- galactoceramide, monophosphoryl-lipid A (MPLA), Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx), IMO-2055 (EMD1201081), TMX-101 (imiquimod), MGN1703 (a TLR9 agonist), Ribi (a TLR4 agonist), G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A), GLA-LSQ (a Glucopyranosyl lipid adjuvant in a liposomal formulation with QS21), Entolimod (a derivative of Salmonella flagellin also known as CBLB502), Hiltonol (a TLR3 agonist), and Imiquimod), and / or inhibitors of heat-shock protein 90 (Hsp90), such as 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0158] In particular embodiments a squalene-based adjuvant can be used. Squalene is part of the group of molecules known as triterpenes, which are all hydrocarbons with 30 carbon molecules. Squalene can be derived from certain plant sources, such as rice bran, wheat germ, amaranth seeds, and olives, as well as from animal sources, such as shark liver oil. In particular embodiments, the squalene-based adjuvant is MF59® (Novartis, Basel, Switzerland). An example of a squalene-based adjuvant that is similar to MF59® but is designed for preclinical research use is Addavax™ (InvivoGen, San Diego, CA). MF59 has been FDA approved for use in an influenza vaccine, and studies indicate that it is safe for use during pregnancy (Tsai T, et al. Vaccine.2010. 17:28(7):1877-80; Heikkinen T, et al. American Journal of Obstetrics and Gynecology. 2012. 207(3):177). In particular embodiments, squalene-based adjuvants can include 0.1% -20% (v / v) squalene oil. In particular embodiments, squalene-based adjuvants can include 5%(v / v) squalene oil.

[0159] In particular embodiments the adjuvant alum can be used. Alum refers to a family of salts that contain two sulfate groups, a monovalent cation, and a trivalent metal, such as aluminum or chromium. Alum is an FDA approved adjuvant. In particular embodiments, vaccines can include alum in the amounts of 1-1000 µg / dose or 0.1 mg-10 mg / dose.

[0160] In particular embodiments, the adjuvant Vaxfectin® (Vical, Inc., San Diego, CA) can be used. Vaxfectin® is a cationic lipid based adjuvant.

[0161] In particular embodiments, one or more STING agonists are used as a vaccine adjuvant. "STING" is an abbreviation of "stimulator of interferon genes", which is also known as "endoplasmic reticulum interferon stimulator (ERIS)", "mediator of IRF3 activation (MITA)", "MPYS" or "transmembrane protein 173 (TM173)". STING is a transmembrane receptor protein and is encoded by the gene TMEM173 in human. Activation of STING leads to production of TypeF053-0192PCT / 23-207-WO-PCT I interferons (e.g., IFN-α and IFN-β), via the IRF3 (interferon regulatory factor 3) pathway; and to production of pro -inflammatory cytokines (e.g., TNF-α and IL-Ιβ), via the NF-κΒ pathway and / or the NLRP3 inflammasome. Particular examples of STING agonists include c-AIMP; (3’,2’)c-AIMP; (2’,2’)c-AIMP; (2’,3’)c-AIMP; c-AIMP(S); c-(dAMP-dIMP); c-(dAMP-2’FdIMP); c-(2’FdAMP- 2’FdIMP); (2’,3’)c-(AMP-2’FdIMP); c-[2’FdAMP(S)-2’FdIMP(S)]; c-[2’FdAMP(S)- 2’FdIMP(S)](POM)2; and DMXAA. Additional examples of STING agonists are described in WO2016 / 145102.

[0162] Other immune stimulants can also be used as vaccine adjuvants. Additional exemplary small molecule immune stimulants include TGF-β inhibitors, SHP-inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Exemplary siRNA capable of down-regulating immune-suppressive signals or oncogenic pathways (such as kras) can be used and any plasmid DNA (such as minicircle DNA) encoding immune-stimulatory proteins can also be used.

[0163] Vaccine adjuvants can be formulated for administration separately from a composition including an antigen or can be formulated in combination with an antigen.

[0164] (viii) Kits. Combinations of components to practice the immunization schedules disclosed herein can also be provided as kits. Kits can include containers including one or more antigens, engineered antigens, multimerized antigens, SOSIP antigens, multimerizing agents, lipids, particles, and / or vaccine adjuvants described herein formulated individually, or in various combinations.

[0165] Kits can also include a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. The notice may state that the provided active ingredients can be administered to a subject. The kits can include further instructions for using the kit, for example, instructions regarding preparation of components for administration; proper disposal of related waste; and the like. The instructions can be in the form of printed instructions provided within the kit or the instructions can be printed on a portion of the kit itself. Instructions may be in the form of a sheet, pamphlet, brochure, CD- Rom, or computer-readable device, or can provide directions to instructions at a remote location, such as a website. In particular embodiments, kits can also include some or all of the necessary medical supplies needed to use the kit effectively, such as syringes, ampules, tubing, facemask, an injection cap, sponges, sterile adhesive strips, Chloraprep, gloves, and the like. Variations in contents of any of the kits described herein can be made. The instructions of the kit will direct use of the active ingredients to effectuate a new clinical use described herein.

[0166] (ix) Methods of Use. Once formed, the compositions are used in immunization schedules.F053-0192PCT / 23-207-WO-PCT In particular embodiments, the compositions elicit antibodies that recognize a full length Env protein. In particular embodiments, the compositions find use in the prevention of disease. “Prevention” refers to preventing as well as reducing the occurrence or severity of a targeted pathological condition or disorder, such as HIV. "Subjects" include those at risk of contracting HIV (such as due to environmental factors, behavioral factors, occupational exposure, social determinants, and travel history), those seeking protection against HIV infection (such as immunocompromised subjects), and any subject who may benefit from immunization against HIV. Thus, in various exemplary embodiments, a subject can be a human subject. Other types of subjects include appropriate research animals.

[0167] In particular embodiments, compositions can be administered to a subject in a therapeutically effective amount. A "therapeutically effective amount” is an amount sufficient to produce a desired physiological effect and / or an amount capable of achieving a desired result, particularly for prevention of a disorder or disease condition, including preventing or delaying the onset of the disorder or disease or minimizing the severity of one or more symptom of the disorder or disease. Therapeutically effective amounts can provide immunization against a disease.

[0168] Particular uses of the compositions include use as vaccines. Vaccines increase the immunity of a subject against a particular disease. Therefore, "HIV vaccine" can refer to a treatment that increases the immunity of a subject against HIV. In particular embodiments, the vaccine initiates the elicitation of antibodies that can bind a full length Env in subjects not currently infected with HIV.

[0169] In particular embodiments, an HIV vaccine is a therapeutically effective composition including one or more antigen or engineered, multimerized, or SOSIP antigen disclosed herein that induce an immune response in a subject against HIV. The skilled artisan will appreciate that the immune system generally is capable of producing an innate immune response and an adaptive immune response. An innate immune response generally can be characterized as not being substantially antigen specific and / or not generating immune memory. An adaptive immune response can be characterized as being substantially antigen specific, maturing over time (e.g., increasing affinity and / or avidity for antigen), and in general can produce immunologic memory. Even though these and other functional distinctions between innate and adaptive immunity can be discerned, the skilled artisan will appreciate that the innate and adaptive immune systems can be integrated and therefore can act in concert.

[0170] "Immune response" refers to a response of the immune system to an antigen disclosed herein. In various exemplary embodiments, an immune response to an antigen can be an innate and / or adaptive response. In some embodiments, an adaptive immune response can be aF053-0192PCT / 23-207-WO-PCT "primary immune response" which refers to an immune response occurring on the first exposure of a "naive" subject to an engineered antigen that binds a glBCR (e.g., a glVRC01 BCR). For example, in the case of a primary antibody response, after a lag or latent period of from 3 to 14 days depending on, for example, the composition, dose, and subject, gl antibodies to the engineered antigen can be produced. Generally, IgM production lasts for several days followed by IgG production and the IgM response can decrease. Antibody production can terminate after several weeks but memory cells can be produced. In some embodiments, an adaptive immune response can be a "secondary immune response", "anamnestic response," or "booster response" which refer to the immune response occurring after a potential second and subsequent exposure of a subject to a second, third, or fourth administration of an engineered antigen.

[0171] In particular embodiments, an immune response against HIV will include antibody production against the gp120 domain of an engineered antigen.

[0172] Antibodies that result from the disclosed immunization schedules are induced by sequential administration of antigen according to the methods disclosed herein. In some embodiments, an antibody can bind to a gp120 domain of an engineered antigen. In some embodiments, an elicited antibody binds to gp120 (i.e., an etiologic agent of HIV). Without being bound by theory, in some embodiments, the binding of an antibody can substantially neutralize or inactivate autologous HIV gp120. Thus, antibodies are capable of reducing or eliminating a pathologic effect of HIV. That is, the binding of antibodies to gp120 of HIV may decrease or eliminate HIV infectivity and / or virulence factor activity, including replication, synthesis, and / or toxicity. In particular embodiments, at least a 25% decrease of one of these parameters is required to determine that a dose provides a therapeutically effective amount.

[0173] The actual dose amount of antigen administered to a particular subject as well as the timing between sequential antigen administrations can be determined and adjusted by a physician, veterinarian, or researcher taking into account parameters such as physical and physiological factors including target, body weight, pre-existing conditions, idiopathy of the subject, and route of administration.

[0174] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Exemplary doses include 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 or 250 µg / kg body mass or mg / kg body mass although higher and / or lower doses can be used. The number of doses that can be administered as a function of time can be from 1, 2, 3, 4 or 5 doses over up to 25 weeks but canF053-0192PCT / 23-207-WO-PCT be increased or decreased depending at least in part on the immune status of a subject.

[0175] The engineered antigen can be prepared by expressing polynucleotide sequences in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells using standard molecular biology methods (e.g., Sambrook et al.1989, Molecular Cloning a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; incorporated herein by reference).

[0176] (x) Exemplary Embodiments. The Exemplary Embodiments below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure. 1. A method of eliciting antibodies that bind human immunodeficiency virus (HIV) envelope protein (Env) in a subject, the method including administering to the subject an immunization schedule that includes administration of 3 to 5 antigens over a course of 9-25 weeks, wherein a first administered antigen binds a germline (gl)VRC01 B cell receptor (BCR). 2. The method of embodiment 1, wherein the first administered antigen includes an engineered and multimerized 426c core. 3. The method of embodiment 1 or 2, wherein a last administered antigen includes a group M SOSIP. 4. The method of any of embodiments 1-3, wherein a second administered antigen includes a multimerized HxB2 core. 5. The method of any of embodiments 1-3, wherein a second administered antigen includes a chimeric 426c SOSIP (chi-426c SOSIP) or a wild-type 426c core SOSIP 426c WT SOSIP). 6. The method of any of embodiments 1-3, wherein a second administered antigen includes a chi-426c SOSIP. 7. The method of any of embodiments 1-6, wherein a third administered antigen includes a chi- 426c SOSIP or a 426c WT SOSIP. 8. The method of any of embodiments 1-6, wherein a third administered antigen includes a chi- 426c SOSIP. 9. The method of any of embodiments 1-8, wherein the immunization schedule includes two administrations of a group M SOSIP. 10. The method of any of embodiments 1-9, wherein the immunization schedule includes administration of a multimerized HxB2 core and a group M SOSIP. 11. The method of any of embodiments 1-10, wherein the immunization schedule includesF053-0192PCT / 23-207-WO-PCT administration of a 426c core SOSIP and a group M SOSIP. The method of any of embodiments 1-11, wherein the immunization schedule includes administration of a multimerized HxB2 core, a 426c SOSIP, and a group M SOSIP. The method of any of embodiments 1-4, wherein the immunization schedule includes a second antigen including a multimerized HxB2 core, and a third antigen including a group M SOSIP. The method of any of embodiments 1-3, wherein the immunization schedule includes a second antigen including a 426c SOSIP, and a third antigen including a group M SOSIP. The method of any of embodiments 1-4, wherein the immunization schedule includes a second antigen including a multimerized HxB2 core, a third antigen including a group M SOSIP, and and a fourth antigen including a group M SOSIP. The method of any of embodiments 1-3, wherein the immunization schedule includes a second antigen including a 426c SOSIP, a third antigen including a group M SOSIP, and and a fourth antigen including a group M SOSIP. The method of any of embodiments 1-4, wherein the immunization schedule includes a second antigen including a multimerized HxB2 core, a third antigen including a 426c SOSIP, and a fourth antigen including a group M SOSIP. The method of any of embodiments 1-4, wherein the immunization schedule includes a second antigen including a multimerized HxB2 core, a third antigen including a 426c SOSIP, a fourth antigen including a group M SOSIP, and and a fifth antigen including a group M SOSIP. The method of any of embodiments 5-12, 14, or 16-18, wherein the 426c SOSIP is a chi-426c SOSIP or a 426c WT SOSIP. The method of any of embodiments 5-12, 14, or 16-19, wherein the 426c SOSIP is a chi-426c SOSIP. The method of any of embodiments 12-20, wherein the immunization schedule does not include administration of any other antigens. The method of any of embodiments 1-21, wherein the immunization schedule is complete within 21 weeks, within 20 weeks, or within 14 weeks.F053-0192PCT / 23-207-WO-PCT The method of any of embodiments 2-22, wherein the engineered and multimerized 426c core includes: (i) mutations at one or more of: N460D, N463D, S278R, G471S, V65C, and S115C; (ii) removal of the V1 loop and removal of the V2 loop; (iii) replacement of the V3 loop with a flexible linker; and (iv) an N-terminal truncation; wherein the engineered and multimerized 426c core does not have a mutation at position 276. The method of any of embodiments 2-23, wherein the engineered and multimerized 426c core includes mutations at N460D, N463D, S278R, G471S, V65C, and S115C. The method of embodiment 23 or 24, wherein the N-terminal truncation is directly before residue 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 or 39. The method of embodiment 23 or 24, wherein the N-terminal truncation is directly before residue 44. The method of any of embodiments 2-26, wherein the engineered and multimerized 426c core further includes a C-terminal truncation directly after residue 499, 498, 497, 496, 495, 494, 493, 492, 491, 490 or 389. The method of embodiment 27, wherein the C-terminal truncation is directly after residue 494. The method of any of embodiments 23-28, wherein the flexible linker includes a sequence as set forth in any one of SEQ ID NOs: 2-72. The method of any of embodiments 23-29, wherein the V3 loop includes residues 296-331. The method of any of embodiments 23-30, wherein removal of the V1 loop includes removal of residues 131-152 and / or removal of the V2 loop includes removal of residues 161-196. The method of any of embodiments 23-30, wherein removal of the V1 loop and removal of the V2 loop includes removal of residues 123-196. The method of any of embodiments 2-32, wherein the engineered and multimerized 426c core is multimerized with a C4b multimerization domain. The method of embodiment 33, wherein the C4b multimerization domain includes a sequence as set forth in any one of SEQ ID NOs: 122-154 or a sequence having at least 98% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 122-154. The method of any of embodiments 2-34, wherein the engineered and multimerized 426c core includes a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 1. The method of any of embodiments 2-35, wherein the engineered and multimerized 426c core includes a 426c core, a linker, and C4b multimerization domain. The method of embodiment 36, wherein the engineered and multimerized 426c core includes a sequence as set forth in SEQ ID NO: 159 or SEQ ID NO: 160 or a sequence having at leastF053-0192PCT / 23-207-WO-PCT 98% sequence identity to the sequence as set forth in SEQ ID NO: 159 or SEQ ID NO: 160. The method of embodiment 4, wherein the multimerized HxB2 core is a wild-type HxB2 core. The method of embodiment 4-38, wherein the multimerized HxB2 core is multimerized with C4b. The method of embodiment 38 or 39, wherein the wild-type HxB2 core includes a sequence as set forth in SEQ ID NO: 161 or 162 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 161 or 162. The method of any of embodiments 4-40, wherein the multimerized HxB2 core includes a HxB2 core, a linker, and C4b multimerization domain. The method of embodiment 41, wherein the multimerized HxB2 core includes a sequence as set forth in SEQ ID NO: 163 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 163. The method of any of embodiments 5-12, 19-37, or 39-42, wherein the chi-426c SOSIP includes a sequence as set forth in SEQ ID NO: 164 or 165 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 164 or 165. The method of any of embodiments 5-12, 19-37, or 39-43, wherein the chi-426c SOSIP is encoded by a sequence as set forth in SEQ ID NO: 166 or 167 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 166 or 167. The method of any of embodiments 5, 7-12, 19-37, or 39-44, wherein the 426c WT SOSIP includes a sequence as set forth in SEQ ID NO: 172 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 172. The method of any of embodiments 3-45, wherein the group M SOSIP includes a sequence as set forth in SEQ ID NO: 168 or 169 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 168 or 169. The method of any of embodiments 3-46, wherein the group M SOSIP is encoded by a sequence as set forth in SEQ ID NO: 170 or 171 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 170 or 171. The method of any of embodiments 1-47, wherein a time between the first antigen administration and a second antigen administration is 3 to 7 weeks. The method of any of embodiments 1-47, wherein a time between the first antigen administration and a second antigen administration is 4 or 6 weeks. The method of any of embodiments 1-47, wherein a time between a second antigen administration and a third antigen administration is 3 to 7 weeks. The method of any of embodiments 1-47, wherein a time between a second antigenF053-0192PCT / 23-207-WO-PCT administration and a third antigen administration is 4 or 6 weeks. The method of any of embodiments 1-47, wherein a time between a third antigen administration and a fourth antigen administration is 3 to 7 weeks. The method of any of embodiments 1-47, wherein a time between a third antigen administration and a fourth antigen administration is 4 or 6 weeks. The method of any of embodiments 1-47, wherein at least one of the 3-5 antigen is administered as a recombinant protein. The method of any of embodiments 1-47, wherein at least one of the 3-5 antigen is administered as a nucleic acid. The method of any of embodiments 1-47, wherein three antigen are administered as recombinant protein and two antigens are administered as nucleic acids. The method of any of embodiments 1-47, wherein the first two antigens are administered as nucleic acids and the third, fourth, and fifth antigens are administered as nucleic acids. The method of any of embodiments 1-47, wherein the first three antigens are administered as recombinant proteins and the fourth and fifth antigens are administered as nucleic acids. The method of any of embodiments 1-47, wherein all antigens of the immunization schedule are administered as recombinant proteins. The method of any of embodiments 1-47, wherein all antigens of the immunization schedule are administered as nucleic acids. The method of embodiment 60, wherein the nucleic acids includes DNA or RNA. The method of embodiment 61, wherein the RNA includes self-amplifying RNA. The method of any of embodiments 1-62, wherein at least one antigen is encapsulated within a nanoparticle. The method of embodiment 63, wherein the nanoparticle is a lipid nanoparticle. The method of embodiment 64, wherein the lipid nanoparticle includes 2-(Di((9Z,12Z)- octadeca-9,12-dien-1-yl)amino)ethan-1-ol, Dinonyl 8,8'-((2- hydroxyethyl)azanediyl)dioctanoate, Nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien- 1-yl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1- yl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8- oxooctyl)amino)octanoate, Di(heptadecan-9-yl) 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate, Heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4-oxobutyl)amino)octanoate, Henicosan- 11-yl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate, or 8-((8-F053-0192PCT / 23-207-WO-PCT (Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoic acid. The method of embodiment 64, wherein the lipid nanoparticle includes Heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate. The method of any of embodiments 1-66, further including administering an adjuvant to the subject. The method of embodiment 67, wherein the administering of the adjuvant is with the first antigen administration or the second antigen administration. The method of embodiment 67 or 68, wherein the administering of the adjuvant is with a third antigen administration or a fourth antigen administration. The method of any of embodiments 67-69, wherein the adjuvant includes one or more of PolyIC, Adjuplex, Alum, Ribi, or GLA-LSQ. A kit including (i) instructions for an immunization schedule that instructs administration of 3 to 5 antigens to a subject over a course of 9-25 weeks, and (ii) a first antigen that binds a germline (gl)VRC01 B cell receptor (BCR). The kit of embodiment 71, wherein the first antigen includes an engineered and multimerized 426c core. The kit of embodiment 71 or 72, further including a group M SOSIP. The kit of embodiment 73, wherein the instructions instruct that the group M SOSIP be administered as a last antigen within the immunization schedule. The kit of any of embodiments 71-74, further including a multimerized HxB2 core. The kit of embodiment 75, wherein the instructions instruct that the multimerized HxB2 core be administered second within the immunization schedule. The kit of any of embodiments 71-76, further including a 426c SOSIP. The kit of embodiment 77, wherein the 426c SOSIP is a chimeric 426c SOSIP (chi-426c SOSIP)_ or a wild-type 426c SOSIP (426c WT SOSIP). The kit of embodiment 77, wherein the 426c SOSIP is a chi-426c SOSIP. The kit of any of embodiments 77-79, wherein the instructions instruct that the 426c SOSIP be administered second within the immunization schedule. The kit of any of embodiments 77-80, wherein the instructions instruct that the 426c SOSIP be administered third within the immunization schedule. The kit of any of embodiments 73-81, wherein the instructions instruct two administrations of the group M SOSIP. The kit of any of embodiments 71-82, wherein the instructions instruct administration of a multimerized HxB2 core and a group M SOSIP.F053-0192PCT / 23-207-WO-PCT The kit of any of embodiments 71-83, wherein the instructions instruct administration of a 426c SOSIP and a group M SOSIP. The kit of any of embodiments 71-84, wherein the instructions instruct administration of a multimerized HxB2 core, a 426c SOSIP, and a group M SOSIP. The kit of any of embodiments 71-79, wherein the instructions instruct administration of a second antigen including a multimerized HxB2 core, and a third antigen including a group M SOSIP. The kit of any of embodiments 71-75, wherein the instructions instruct administration of a second antigen including a 426c SOSIP, and a third antigen including a group M SOSIP. The kit of any of embodiments 71-79, wherein the instructions instruct administration of a second antigen including a multimerized HxB2 core, a third antigen including a group M SOSIP, and and a fourth antigen including a group M SOSIP. The kit of any of embodiments 71-75, wherein the instructions instruct administration of a second antigen including a 426c SOSIP, a third antigen including a group M SOSIP, and and a fourth antigen including a group M SOSIP. The kit of any of embodiments 71-79, wherein the instructions instruct administration of a second antigen including a multimerized HxB2 core, a third antigen including a 426c SOSIP, and a fourth antigen including a group M SOSIP. The kit of any of embodiments 71-79, wherein the instructions instruct administration of a second antigen including a multimerized HxB2 core, a third antigen including a 426c SOSIP, a fourth antigen including a group M SOSIP, and and a fifth antigen including a group M SOSIP. The kit of any of embodiments 71-91, wherein the instructions do not instruct administration of any other antigens. The kit of any of embodiments 72-92, wherein the engineered and multimerized 426c core includes: (i) mutations at one or more of: N460D, N463D, S278R, G471S, V65C, and S115C; (ii) removal of the V1 loop and removal of the V2 loop; (iii) replacement of the V3 loop with a flexible linker; and (iv) an N-terminal truncation; wherein the HIV Env does not have a mutation at position 276.F053-0192PCT / 23-207-WO-PCT The kit of any of embodiments 72-93, wherein the engineered and multimerized 426c core includes mutations at N460D, N463D, S278R, G471S, V65C, and S115C. The kit of embodiment 93 or 94, wherein the N-terminal truncation is directly before residue 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 or 39. The kit of embodiment 93 or 94, wherein the N-terminal truncation is directly before residue 44. The kit of any of embodiments 72-96, wherein the engineered and multimerized 426c core further includes a C-terminal truncation directly after residue 499, 498, 497, 496, 495, 494, 493, 492, 491, 490 or 389. The kit of embodiment 97, wherein the C-terminal truncation is directly after residue 494. The kit of any of embodiments 93-98, wherein the flexible linker includes a sequence as set forth in any one of SEQ ID NOs: 2-72. . The kit of any of embodiments 93-99, wherein the V3 loop includes residues 296-331.. The kit of any of embodiments 93-100, wherein removal of the V1 loop includes removal of residues 131-152 and / or removal of the V2 loop includes removal of residues 161-196.. The kit of any of embodiments 93-100, wherein removal of the V1 loop and removal of the V2 loop includes removal of residues 123-196. . The kit of any of embodiments 72-102, wherein the engineered and multimerized 426c core is multimerized with a C4b multimerization domain. . The kit of embodiment 103, wherein the C4b multimerization domain includes a sequence as set forth in any one of SEQ ID NOs: 122-154 or a sequence having at least 98% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 122-154. . The kit of any of embodiments 72-104, wherein the engineered and multimerized 426c core includes a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 1. . The kit of any of embodiments 72-105, wherein the engineered and multimerized 426c core includes a 426c core, a linker, and C4b multimerization domain. . The kit of embodiment 106, wherein the engineered and multimerized 426c core includes a sequence as set forth in SEQ ID NO: 159 or SEQ ID NO: 160 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 159 or SEQ ID NO: 160.. The kit of any of embodiments 75-107, wherein the multimerized HxB2 core is a wild-type HxB2 core. . The kit of any of embodiments 75-108, wherein the multimerized HxB2 core is multimerized with a C4b multimerization domain.F053-0192PCT / 23-207-WO-PCT . The kit of embodiment 108 or 109, wherein the wild-type HxB2 core includes a sequence as set forth in SEQ ID NO: 161 or 162 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 161 or 162. . The kit of any of embodiments 75-110, wherein the multimerized HxB2 core includes a HxB2 core, a linker, and C4b multimerization domain. . The kit of embodiment 111, wherein the multimerized HxB2 core includes a sequence as set forth in SEQ ID NO: 163 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 163. . The kit of any of embodiments 78-86, 88, or 90-112, wherein the chi-426c SOSIP includes a sequence as set forth in SEQ ID NO: 164 or 165 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 164 or 165. . The kit of any of embodiments 78-86, 88, or 90-113, wherein the chi-426c SOSIP is encoded by a sequence as set forth in SEQ ID NO: 166 or 167 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 166 or 167. . The kit of any of embodiments 78, 80-86, 88, or 90-114, wherein the 426c WT SOSIP includes a sequence as set forth in SEQ ID NO: 172 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 172. . The kit of any of embodiments 73-115, wherein the group M SOSIP includes a sequence as set forth in SEQ ID NO: 168 or 169 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 168 or 169. . The kit of any of embodiments 73-116, wherein the group M SOSIP is encoded by a sequence as set forth in SEQ ID NO: 170 or 171 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 170 or 171. . The kit of any of embodiments 71-117, wherein an instructed time between the first antigen administration and the second antigen administration is 3 to 7 weeks. . The kit of any of embodiments 71-117, wherein an instructed time between the first antigen administration and a second antigen administration is 4 or 6 weeks. . The kit of any of embodiments 71-117, wherein an instructed time between a second antigen administration and a third antigen administration is 3 to 7 weeks. . The kit of any of embodiments 71-117, wherein an instructed time between a second antigen administration and a third antigen administration is 4 or 6 weeks. . The kit of any of embodiments 71-117, wherein an instructed time between a third antigen administration and a fourth antigen administration is 3 to 7 weeks. . The kit of any of embodiments 71-117, wherein an instructed time between a third antigenF053-0192PCT / 23-207-WO-PCT administration and a fourth antigen administration is 4 or 6 weeks. . The kit of any of embodiments 71-117, wherein at least one of the 3-5 antigen is a recombinant protein. . The kit of any of embodiments 71-117, wherein at least one of the 3-5 antigen is a nucleic acid. . The kit of embodiment 125, wherein the nucleic acid includes DNA or RNA. . The kit of embodiment 126, wherein the RNA includes self-amplifying RNA. . The kit of any of embodiments 71-127, wherein three antigen are recombinant protein and two antigen are nucleic acid. . The kit of any of embodiments 71-127, wherein the first two antigen are recombinant protein and the third, fourth, and fifth antigen are nucleic acid. . The kit of any of embodiments 71-127, wherein the first three antigen are recombinant protein and the fourth and fifth antigen are nucleic acid. . The kit of any of embodiments 71-124, wherein all antigen are recombinant protein.. The kit of any of embodiments 71-123, wherein all antigen are nucleic acid. . The kit of any of embodiments 71-132, wherein at least one antigen is encapsulated within a nanoparticle. . The kit of embodiment 133, wherein the nanoparticle is a lipid nanoparticle. . The kit of embodiment 134, wherein the lipid nanoparticle includes 2-(Di((9Z,12Z)- octadeca-9,12-dien-1-yl)amino)ethan-1-ol, Dinonyl 8,8'-((2- hydroxyethyl)azanediyl)dioctanoate, Nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien- 1-yl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1- yl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8- oxooctyl)amino)octanoate, Di(heptadecan-9-yl) 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate, Heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4-oxobutyl)amino)octanoate, Henicosan- 11-yl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate, or 8-((8- (Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoic acid. . The kit of embodiment 134, wherein the lipid nanoparticle includes Heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate. . The kit of any of embodiments 71-136, further including an adjuvant. . The kit of embodiment 137, wherein the adjuvant includes one or more of PolyIC, Adjuplex, Alum, Ribi, or GLA-LSQ.F053-0192PCT / 23-207-WO-PCT

[0177] (xii) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0178] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0179] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non- polar): Proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0180] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol.157(1), 105-32).F053-0192PCT / 23-207-WO-PCT Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glutamate (−3.5); Gln (−3.5); aspartate (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

[0181] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0182] As detailed in US 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (−0.4); Pro (−0.5±1); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr (−2.3); Phe (−2.5); Trp (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0183] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0184] As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically-significant degree.

[0185] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0186] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequenceF053-0192PCT / 23-207-WO-PCT relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0187] Variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 µg / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCl;F053-0192PCT / 23-207-WO-PCT 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 µg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0188] "Specifically binds" refers to an association of a binding domain (of, for example, a CAR binding domain or a nanoparticle selected cell targeting ligand) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M-1, while not significantly associating with any other molecules or components in a relevant environment sample. “Specifically binds” is also referred to as “binds” herein. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Ka of at least 107M-1, at least 108M-1, at least 109M-1, at least 1010M-1, at least 1011M-1, at least 1012M-1, or at least 1013M-1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 107M-1, up to 106M-1, up to 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5M to 10-13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off- rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® (GE Healthcare, United States) analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci.51:660; and US 5,283,173, US 5,468,614, or the equivalent).

[0189] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., GreenF053-0192PCT / 23-207-WO-PCT and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes- Davis and R. I. Freshney, eds. Freshney’s Culture of Animal Cells 8thEdition (2021).

[0190] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in ability of elicited antibodies to neutralize HIV.

[0191] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value. In particular embodiments, the residue numbering of mutation and deletion positions of Env is precise, rather than approximate.

[0192] Notwithstanding that the numerical ranges and parameters setting forth the broad scopeF053-0192PCT / 23-207-WO-PCT of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0193] The terms “a,” “an,” “the” and similar referents used 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. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual 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 otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0194] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0195] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than 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.

[0196] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein).F053-0192PCT / 23-207-WO-PCT Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0197] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0198] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0199] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

F053-0192PCT / 23-207-WO-PCT CLAIMS What is claimed is:

1. A method of eliciting antibodies that bind human immunodeficiency virus (HIV) envelope protein (Env) in a subject, the method comprising administering to the subject an immunization schedule that comprises: a first antigen comprising engineered and multimerized 426c core, a second antigen comprising a multimerized HxB2 core, a third antigen comprising a chimeric 426c SOSIP (chi-426c SOSIP), and a fourth antigen comprising a group M SOSIP.

2. The method of claim 1, wherein the second antigen is administered 4 weeks after the first antigen is administered, the third antigen is administered 10 weeks after the first antigen is administered, and the fourth antigen is administered 14 weeks after the first antigen is administered.

3. A method of eliciting antibodies that bind human immunodeficiency virus (HIV) envelope protein (Env) in a subject, the method comprising administering to the subject an immunization schedule that comprises administration of 3 to 5 antigens over a course of 9- 25 weeks, wherein a first administered antigen binds a germline (gl)VRC01 B cell receptor (BCR).

4. The method of claim 3, wherein the first administered antigen comprises an engineered and multimerized 426c core.

5. The method of claim 3, wherein a last administered antigen comprises a group M SOSIP.

6. The method of claim 3, wherein a second administered antigen comprises a multimerized HxB2 core.

7. The method of claim 3, wherein a second administered antigen comprises a chimeric 426c SOSIP (chi-426c SOSIP) or a wild-type 426c SOSIP (426c WT SOSIP).

8. The method of claim 3, wherein a second administered antigen comprises a chi-426c SOSIP.

9. The method of claim 3, wherein a third administered antigen comprises a chi-426c SOSIP or a 426c WT SOSIP.

10. The method of claim 3, wherein a third administered antigen comprises a chi-426c SOSIP.

11. The method of claim 3, wherein the immunization schedule comprises two administrations of a group M SOSIP.

12. The method of claim 3, wherein the immunization schedule comprises administration of a multimerized HxB2 core and a group M SOSIP.

13. The method of claim 3, wherein the immunization schedule comprises administration of aF053-0192PCT / 23-207-WO-PCT 426c SOSIP and a group M SOSIP.

14. The method of claim 3, wherein the immunization schedule comprises administration of a multimerized HxB2 core, a 426c SOSIP, and a group M SOSIP.

15. The method of claim 3, wherein the immunization schedule comprises a second antigen comprising a multimerized HxB2 core, and a third antigen comprising a group M SOSIP.

16. The method of claim 3, wherein the immunization schedule comprises a second antigen comprising a 426c SOSIP, and a third antigen comprising a group M SOSIP.

17. The method of claim 3, wherein the immunization schedule comprises a second antigen comprising a multimerized HxB2 core, a third antigen comprising a group M SOSIP, and and a fourth antigen comprising a group M SOSIP.

18. The method of claim 3, wherein the immunization schedule comprises a second antigen comprising a 426c SOSIP, a third antigen comprising a group M SOSIP, and and a fourth antigen comprising a group M SOSIP.

19. The method of claim 3, wherein the immunization schedule comprises a second antigen comprising a multimerized HxB2 core, a third antigen comprising a 426c SOSIP, and a fourth antigen comprising a group M SOSIP.

20. The method of claim 3, wherein the immunization schedule comprises a second antigen comprising a multimerized HxB2 core, a third antigen comprising a 426c SOSIP, a fourth antigen comprising a group M SOSIP, and and a fifth antigen comprising a group M SOSIP.

21. The method of claim 14, wherein the 426c SOSIP is a chi-426c SOSIP or a wild-type (WT) 426c SOSIP.

22. The method of claim 14, wherein the 426c SOSIP is a chi-426c SOSIP.

23. The method of claim 14, wherein the immunization schedule does not comprise administration of any other antigens.

24. The method of claim 3, wherein the immunization schedule is complete within 21 weeks, within 20 weeks, or within 14 weeks.

25. The method of claim 4, wherein the engineered and multimerized 426c core comprises: (i)F053-0192PCT / 23-207-WO-PCT mutations at one or more of: N460D, N463D, S278R, G471S, V65C, and S115C; (ii) removal of a V1 loop and removal of a V2 loop; (iii) replacement of a V3 loop with a flexible linker; and (iv) an N-terminal truncation; wherein the engineered and multimerized 426c core does not have a mutation at position 276.

26. The method of claim 4, wherein the engineered and multimerized 426c core comprises mutations at N460D, N463D, S278R, G471S, V65C, and S115C.

27. The method of claim 25, wherein the N-terminal truncation is directly before residue 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 or 39.

28. The method of claim 25, wherein the N-terminal truncation is directly before residue 44.

29. The method of claim 4, wherein the engineered and multimerized 426c core further comprises a C-terminal truncation directly after residue 499, 498, 497, 496, 495, 494, 493, 492, 491, 490 or 389.

30. The method of claim 29, wherein the C-terminal truncation is directly after residue 494.

31. The method of claim 25, wherein the flexible linker comprises a sequence as set forth in any one of SEQ ID NOs: 2-72.

32. The method of claim 25, wherein the V3 loop comprises residues 296-331.

33. The method of claim 25, wherein removal of the V1 loop comprises removal of residues 131- 152 and / or removal of the V2 loop comprises removal of residues 161-196.

34. The method of claim 25, wherein removal of the V1 loop and removal of the V2 loop comprises removal of residues 123-196.

35. The method of claim 4, wherein the engineered and multimerized 426c core is multimerized with a C4b multimerization domain.

36. The method of claim 35, wherein the C4b multimerization domain comprises a sequence as set forth in any one of SEQ ID NOs: 122-154 or a sequence having at least 98% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 122-154.

37. The method of claim 4, wherein the engineered and multimerized 426c core comprises a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

1.

38. The method of claim 4, wherein the engineered and multimerized 426c core comprises a 426c core, a linker, and C4b multimerization domain.

39. The method of claim 38, wherein the engineered and multimerized 426c core comprises a sequence as set forth in SEQ ID NO: 159 or SEQ ID NO: 160 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 159 or SEQ ID NO:

160.

40. The method of claim 6, wherein the multimerized HxB2 core is a wild-type HxB2 core.F053-0192PCT / 23-207-WO-PCT 41. The method of claim 6, wherein the multimerized HxB2 core is multimerized with C4b.

42. The method of claim 40, wherein the wild-type HxB2 core comprises a sequence as set forth in SEQ ID NO: 161 or 162 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 161 or 162.

43. The method of claim 6, wherein the multimerized HxB2 core comprises a HxB2 core, a linker, and C4b multimerization domain.

44. The method of claim 43, wherein the multimerized HxB2 core comprises a sequence as set forth in SEQ ID NO: 163 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

163.

45. The method of claim 7, wherein the chi-426c SOSIP comprises a sequence as set forth in SEQ ID NO: 164 or 165 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 164 or 165.

46. The method of claim 7, wherein the chi-426c SOSIP is encoded by a sequence as set forth in SEQ ID NO: 166 or 167 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 166 or 167.

47. The method of claim 7, wherein the 426c WT SOSIP comprises a sequence as set forth in SEQ ID NO: 172 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

172.

48. The method of claim 5, wherein the group M SOSIP comprises a sequence as set forth in SEQ ID NO: 168 or 169 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 168 or 169.

49. The method of claim 5, wherein the group M SOSIP is encoded by a sequence as set forth in SEQ ID NO: 170 or 171 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 170 or 171.

50. The method of claim 3, wherein a time between the first antigen administration and a second antigen administration is 3 to 7 weeks.

51. The method of claim 3, wherein a time between the first antigen administration and a second antigen administration is 4 or 6 weeks.

52. The method of claim 3, wherein a time between a second antigen administration and a third antigen administration is 3 to 7 weeks.

53. The method of claim 3, wherein a time between a second antigen administration and a third antigen administration is 4 or 6 weeks.

54. The method of claim 3, wherein a time between a third antigen administration and a fourth antigen administration is 3 to 7 weeks.F053-0192PCT / 23-207-WO-PCT 55. The method of claim 3, wherein a time between a third antigen administration and a fourth antigen administration is 4 or 6 weeks.

56. The method of claim 3, wherein at least one of the 3-5 antigen is administered as a recombinant protein.

57. The method of claim 3, wherein at least one of the 3-5 antigen is administered as a nucleic acid.

58. The method of claim 3, wherein three antigen are administered as recombinant protein and two antigen are administered as nucleic acid.

59. The method of claim 3, wherein the first two antigens are administered as nucleic acids and the third, fourth, and fifth antigens are administered as nucleic acids.

60. The method of claim 3, wherein the first three antigens are administered as recombinant proteins and the fourth and fifth antigens are administered as nucleic acids.

61. The method of claim 3, wherein all antigens of the immunization schedule are administered as recombinant proteins.

62. The method of claim 3, wherein all antigens of the immunization schedule are administered as nucleic acids.

63. The method of claim 62, wherein the nucleic acid comprises DNA or RNA.

64. The method of claim 63, wherein the RNA comprises self-amplifying RNA.

65. The method of claim 3, wherein at least one antigen is encapsulated within a nanoparticle.

66. The method of claim 65, wherein the nanoparticle is a lipid nanoparticle.

67. The method of claim 66, wherein the lipid nanoparticle comprises 2-(Di((9Z,12Z)-octadeca- 9,12-dien-1-yl)amino)ethan-1-ol, Dinonyl 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate, Nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate, Heptadecan-9-yl 8- ((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate, Heptadecan-9-yl 8- ((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, Di(heptadecan-9-yl) 8,8'-((2- hydroxyethyl)azanediyl)dioctanoate, Heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8- (nonyloxy)-8-oxooctyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4- oxobutyl)amino)octanoate, Henicosan-11-yl 6-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)hexanoate, or 8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2- hydroxyethyl)amino)octanoic acid.

68. The method of claim 66, wherein the lipid nanoparticle comprises Heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate.

69. The method of claim 3, further comprising administering an adjuvant to the subject.F053-0192PCT / 23-207-WO-PCT 70. The method of claim 69, wherein the administering of the adjuvant is with the first antigen administration or the second antigen administration.

71. The method of claim 69, wherein the administering of the adjuvant is with a third antigen administration or a fourth antigen administration.

72. The method of claim 69, wherein the adjuvant comprises one or more of PolyIC, Adjuplex, Alum, Ribi, or GLA-LSQ.

73. A kit comprising (i) instructions for an immunization schedule that instructs administration of 3 to 5 antigens to a subject over a course of 9-25 weeks, and (ii) a first antigen that binds a germline (gl)VRC01 B cell receptor (BCR).

74. The kit of claim 73, wherein the first antigen comprises an engineered and multimerized 426c core.

75. The kit of claim 73, further comprising a group M SOSIP.

76. The kit of claim 75, wherein the instructions instruct that the group M SOSIP be administered as a last antigen within the immunization schedule.

77. The kit of claim 73, further comprising a multimerized HxB2 core.

78. The kit of claim 77, wherein the instructions instruct that the multimerized HxB2 core be administered second within the immunization schedule.

79. The kit of claim 73, further comprising a 426c SOSIP.

80. The kit of claim 79, wherein the 426c SOSIP is a chimeric 426c SOSIP (chi-426c SOSIP) or a wild-type 426c SOSIP (426c WT SOSIP).

81. The kit of claim 79, wherein the 426c SOSIP is a chi-426c SOSIP.

82. The kit of claim 79, wherein the instructions instruct that the 426c SOSIP be administered second within the immunization schedule.

83. The kit of claim 79, wherein the instructions instruct that the 426c SOSIP be administered third within the immunization schedule.

84. The kit of claim 75, wherein the instructions instruct two administrations of the group M SOSIP.

85. The kit of claim 73, wherein the instructions instruct administration of a multimerized HxB2 core and a group M SOSIP.

86. The kit of claim 73, wherein the instructions instruct administration of a 426c SOSIP and a group M SOSIP.

87. The kit of claim 73, wherein the instructions instruct administration of a multimerized HxB2 core, a 426c SOSIP, and a group M SOSIP.

88. The kit of claim 73, wherein the instructions instruct administration ofF053-0192PCT / 23-207-WO-PCT a second antigen comprising a multimerized HxB2 core, and a third antigen comprising a group M SOSIP.

89. The kit of claim 73, wherein the instructions instruct administration of a second antigen comprising a 426c SOSIP, and a third antigen comprising a group M SOSIP.

90. The kit of claim 73, wherein the instructions instruct administration of a second antigen comprising a multimerized HxB2 core, a third antigen comprising a group M SOSIP, and and a fourth antigen comprising a group M SOSIP.

91. The kit of claim 73, wherein the instructions instruct administration of a second antigen comprising a 426c SOSIP, a third antigen comprising a group M SOSIP, and and a fourth antigen comprising a group M SOSIP.

92. The kit of claim 73, wherein the instructions instruct administration of a second antigen comprising a multimerized HxB2 core, a third antigen comprising a 426c SOSIP, and a fourth antigen comprising a group M SOSIP.

93. The kit of claim 73, wherein the instructions instruct administration of a second antigen comprising a multimerized HxB2 core, a third antigen comprising a 426c SOSIP, a fourth antigen comprising a group M SOSIP, and and a fifth antigen comprising a group M SOSIP.

94. The kit of claim 73, wherein the instructions do not instruct administration of any other antigens.

95. The kit of claim 74, wherein the engineered and multimerized 426c core comprises: (i) mutations at one or more of: N460D, N463D, S278R, G471S, V65C, and S115C; (ii) removal of a V1 loop and removal of a V2 loop; (iii) replacement of a V3 loop with a flexible linker; and (iv) an N-terminal truncation; wherein the HIV Env does not have a mutation at position 276.

96. The kit of claim 74, wherein the engineered and multimerized 426c core comprises mutations at N460D, N463D, S278R, G471S, V65C, and S115C.

97. The kit of claim 95, wherein the N-terminal truncation is directly before residue 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 or 39.

98. The kit of claim 95, wherein the N-terminal truncation is directly before residue 44.F053-0192PCT / 23-207-WO-PCT 99. The kit of claim 95, wherein the engineered and multimerized 426c core further comprises a C-terminal truncation directly after residue 499, 498, 497, 496, 495, 494, 493, 492, 491, 490 or 389.

100. The kit of claim 99, wherein the C-terminal truncation is directly after residue 494.

101. The kit of claim 95, wherein the flexible linker comprises a sequence as set forth in any one of SEQ ID NOs: 2-72.

102. The kit of claim 95, wherein the V3 loop comprises residues 296-331.

103. The kit of claim 95, wherein removal of the V1 loop comprises removal of residues 131- 152 and / or removal of the V2 loop comprises removal of residues 161-196.

104. The kit of claim 95, wherein removal of the V1 loop and removal of the V2 loop comprises removal of residues 123-196.

105. The kit of claim 74, wherein the engineered and multimerized 426c core is multimerized with a C4b multimerization domain.

106. The kit of claim 105, wherein the C4b multimerization domain comprises a sequence as set forth in any one of SEQ ID NOs: 122-154 or a sequence having at least 98% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 122-154.

107. The kit of claim 74, wherein the engineered and multimerized 426c core comprises a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

1.

108. The kit of claim 74, wherein the engineered and multimerized 426c core comprises a 426c core, a linker, and C4b multimerization domain.

109. The kit of claim 74, wherein the engineered and multimerized 426c core comprises a sequence as set forth in SEQ ID NO: 159 or SEQ ID NO: 160 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 159 or SEQ ID NO:

160.

110. The kit of claim 77, wherein the multimerized HxB2 core is a wild-type HxB2 core.

111. The kit of claim 77, wherein the multimerized HxB2 core is multimerized with a C4b multimerization domain.

112. The kit of claim 110, wherein the wild-type HxB2 core comprises a sequence as set forth in SEQ ID NO: 161 or 162 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 161 or 162.

113. The kit of claim 77, wherein the multimerized HxB2 core comprises a HxB2 core, a linker, and C4b multimerization domain.

114. The kit of claim 113, wherein the multimerized HxB2 core comprises a sequence as set forth in SEQ ID NO: 163 or a sequence having at least 98% sequence identity to theF053-0192PCT / 23-207-WO-PCT sequence as set forth in SEQ ID NO:

163.

115. The kit of claim 80, wherein the chi-426c SOSIP comprises a sequence as set forth in SEQ ID NO: 164 or 165 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 164 or 165.

116. The kit of claim 80, wherein the chi-426c SOSIP is encoded by a sequence as set forth in SEQ ID NO: 166 or 167 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 166 or 167.

117. The kit of claim 80, wherein the 426c WT SOSIP comprises a sequence as set forth in SEQ ID NO: 172 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

172.

118. The kit of claim 75, wherein the group M SOSIP comprises a sequence as set forth in SEQ ID NO: 168 or 169 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 168 or 169.

119. The kit of claim 75, wherein the group M SOSIP is encoded by a sequence as set forth in SEQ ID NO: 170 or 171 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 170 or 171.

120. The kit of claim 73, wherein an instructed time between the first antigen administration and the second antigen administration is 3 to 7 weeks.

121. The kit of claim 73, wherein an instructed time between the first antigen administration and a second antigen administration is 4 or 6 weeks.

122. The kit of claim 73, wherein an instructed time between a second antigen administration and a third antigen administration is 3 to 7 weeks.

123. The kit of claim 73, wherein an instructed time between a second antigen administration and a third antigen administration is 4 or 6 weeks.

124. The kit of claim 73, wherein an instructed time between a third antigen administration and a fourth antigen administration is 3 to 7 weeks.

125. The kit of claim 73, wherein an instructed time between a third antigen administration and a fourth antigen administration is 4 or 6 weeks.

126. The kit of claim 73, wherein at least one of the 3-5 antigen is a recombinant protein.

127. The kit of claim 73, wherein at least one of the 3-5 antigen is a nucleic acid.

128. The kit of claim 127, wherein the nucleic acid comprises DNA or RNA.

129. The kit of claim 128, wherein the RNA comprises self-amplifying RNA.

130. The kit of claim 73, wherein three antigen are recombinant protein and two antigen are nucleic acid.F053-0192PCT / 23-207-WO-PCT 131. The kit of claim 73, wherein the first two antigen are recombinant protein and the third, fourth, and fifth antigen are nucleic acid.

132. The kit of claim 73, wherein the first three antigen are recombinant protein and the fourth and fifth antigen are nucleic acid.

133. The kit of claim 73, wherein all antigen are recombinant protein.

134. The kit of claim 73, wherein all antigen are nucleic acid.

135. The kit of claim 73, wherein at least one antigen is encapsulated within a nanoparticle.

136. The kit of claim 135, wherein the nanoparticle is a lipid nanoparticle.

137. The kit of claim 136, wherein the lipid nanoparticle comprises 2-(Di((9Z,12Z)-octadeca- 9,12-dien-1-yl)amino)ethan-1-ol, Dinonyl 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate, Nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate, Heptadecan-9-yl 8- ((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate, Heptadecan-9-yl 8- ((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, Di(heptadecan-9-yl) 8,8'-((2- hydroxyethyl)azanediyl)dioctanoate, Heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8- (nonyloxy)-8-oxooctyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4- oxobutyl)amino)octanoate, Henicosan-11-yl 6-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)hexanoate, or 8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2- hydroxyethyl)amino)octanoic acid.

138. The kit of claim 136, wherein the lipid nanoparticle comprises Heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate.

139. The kit of claim 73, further comprising an adjuvant.

140. The kit of claim 139, wherein the adjuvant comprises one or more of PolyIC, Adjuplex, Alum, Ribi, or GLA-LSQ.

Images