Immunogenic trimers

Abstract

The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the viral surface. The Env gpl20 and gp41 subunits mediate receptor binding and membrane fusion. HIV- 1 neutralizing antibodies can be generated following immunization via expressing membrane-bound Env anchored on the cell-surface genetically using the natural HIV gp41 transmembrane (TM) spanning domain. Inventors used "native flexibly linked" (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. The NFL construct was genetically fused to the HIV TM domain using a short linker or restoring the native membrane external proximal region to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles when expressed from plasmid DNA or mRNA. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals.

Description

IMMUNOGENIC TRIMERSRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 662,880, filed June 21, 2024, the entire contents of which are hereby incorporated by reference herein. The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.FIELD OF THE INVENTION

[0002] The present invention relates to HIV Env trimers derived from clinical isolates by membrane-bound array from mRNA lipid nanoparticles. The expressed proteins exhibit a desirable antigenic profile, being recognized by broadly neutralizing antibodies while showing low recognition by non-neutralizing antibodies.BACKGROUND OF THE INVENTION

[0003] The HIV-1 envelope glycoprotein (Env) is the only virally encoded neutralizing determinant on the surface of the virus. The gp!20 and gp41 Env subunits mediate receptor binding and membrane fusion, respectively. These subunits are generated from precursor cleavage of the trimeric gpl60 by cellular furins, liberating the gp41 fusion peptide (FP), required for viral -to-cell membrane fusion and entry. Following furin-mediated activation, the entry-competent trimer of gpl20 and gp41 heterodimers remain associated in a metastable non-covalent manner (1, 2). To elicit Env-directed neutralizing antibodies in vivo directed at the functional spike, investigators in the HIV vaccine field have developed spike mimetics as the first step toward a broadly effective vaccine. To a large extent, the generation of native-like HIV-1 Env spikes has focused on soluble trimers, led by the so-called Env mimics “SOSIP” (3, 4). The gp!40 SOSIP trimers require thesame furin-mediated cleavage for native like conformation along with a helix destabilizing mutation in gp41, I559P (5, 6). An improvement of the SOSIP design is to remove the need for furin cleavage by introduction of a flexible linker between the gpl20 and gpl40 subunits to generate soluble “NFL” trimers (for native flexibly linked)(7, 8). Both SOSIP and NFLs can be produced as recombinant proteins and are being evaluated in the clinic as vaccine candidates. However, production of these recombinant trimers as GMP-grade clinical material is time consuming and expensive. In the likely event that prime-boosting of multiple HIV-1 Env components or strains is required to elicit cross-neutralizing antibodies, then genetic expression should reduce time and cost. The rapid design, development, and vaccination of humans as exemplified by mRNA vaccines expressing multiple variants of membrane-bound SARS-CoV2 spike proteins is strong evidence for such an approach.

[0004] The propensity of Env to generate well-ordered native-like trimeric proteins is helpful for the success of a nucleic acid HIV vaccine as genetic expression in vivo does not allow for the removal of undesired, and non-native forms of Env. These off-target conformers generate ‘immune distracting’ antibody responses against non-native and non-neutralizing determinants. Because the HIV-1 Env is normally expressed with transmembrane anchor points, soluble versions of Env, truncated following residue 664, lack the hydrophobic membrane proximal external region (MPER) and natural transmembrane anchor points and often require internal stabilizing mutations to generate mostly well-ordered trimers (9-15). In addition, ways have been derived to purify the native-like soluble HIV Env trimers from other isoforms using positive or negative selection methods to improve further the homogeneity of the Env candidate immunogens (16, 17). This conformer selection is not possible with membrane-bound trimer expressed by genetic means. It is reasoned that expressing the stabilized 81 NFL trimers anchored to the natural HIV TM might generate a more relevant mimic of the native spike as others have recently demonstrated using alternative HIV Env trimeric platforms (18-21). And, because the NFL Env is stabilized to a degree beyond the natural stability of native Env, the cell-surface expressed trimers might stay conformationally relevant for longer in vivo. Moreover, restoring the membrane occludes the unnaturally exposed protein base soluble trimers display following truncation before the MPER.

[0005] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure relates to a non-naturally occurring protein comprising a native flexibly linked (NFL) trimer of an HIV-1 envelope glycoprotein (Env). The NFL trimer is designed to be cleavage-independent and is linked to a natural HIV transmembrane (TM) domain and a cytoplasmic tail (CT). The protein is further stabilized by a cassette of mutations to enhance homogeneity and stability.

[0007] In some embodiments, the Env comprises a sequence derived from an HIV strain selected from 1086c (Genbank Accession Number FJ444395), JR-FL (Genbank Accession Number U63632); BG505 (Genbank Accession Number DQ208458), or 16055 (Genbank Accession Number EFl 17268).

[0008] In some embodiments, the Env is linked to the HIV TM by a flexible linker.

[0009] In some embodiments, the linker has the sequence (G4S)2.

[0010] In some embodiments, the Env is linked to the HIV TM via a membrane proximal external region (MPER).

[0011] In some embodiments, the MPER extends from residue D664 to K683.

[0012] In some embodiments, the CT is truncated at residue G711 to increase retention of membrane-bound trimeric Env proteins on the cell surface.

[0013] In some embodiments, the protein comprises the amino acid sequence of 1086c NFL.711 (-MPER) as set forth in SEQ ID NO: 1.

[0014] In some embodiments, the protein comprises the amino acid sequence of 1086c NFL.711 (+MPER) as set forth in SEQ ID NO: 2.

[0015] In some embodiments, the protein comprises the amino acid sequence of 16055 NFL.711 as set forth in SEQ ID NO: 3.

[0016] In some embodiments, the protein comprises the amino acid sequence of BG505 NFL.711 as set forth in SEQ ID NO: 4.

[0017] In some embodiments, the protein comprises the amino acid sequence of JR-FL NFL.711 as set forth in SEQ ID NO: 5.

[0018] In some embodiments, the protein has about 95% sequence identity to the amino acid sequence of 1086c NFL.711 (-MPER) as set forth in SEQ ID NO: 1, 1086c NFL.711 (+MPER) as set forth in SEQ ID NO: 2, 16055 NFL.711 as set forth in SEQ ID NO: 3, BG5O5 NFL.711 as set forth in SEQ ID NO: 4, or JR-FL NFL.711 as set forth in SEQ ID NO: 5.

[0019] In some embodiments, the cassette of stabilizing mutations comprises one or more mutations categorized as Trimer Derived (TD) mutations selected from E47D, K49E, V65K, E106T, I165L, E172V, T308R, E429R, R432Q, K500R, L543N, N553S, K588R, or E662A.

[0020] In some embodiments, the cassette of stabilizing mutations comprises one or more mutations categorized as V3 loop stabilizing mutations selected from N302Y or T320M.

[0021] In some embodiments, the cassette of stabilizing mutations comprises one or more mutations categorized as Fusion Peptide (FP) stabilizing mutations selected from F519R or L520R.

[0022] In some embodiments, the cassette of stabilizing mutations comprises one or more mutations categorized as helix-disrupting mutations in gp41 selected from I559P, L568G, T569G, or N636G.

[0023] In some embodiments, the cassette of stabilizing mutations comprises one or more engineered disulfide bonds.

[0024] In some embodiments, the engineered disulfide bond is at positions I201C and A433C.

[0025] In some embodiments, the cassette of stabilizing mutations comprises the addition of an N-glycan.

[0026] In some embodiments, the N-glycan is added at K160N or K334T.

[0027] According to another aspect of the present disclosure, the present disclosure provides for a nucleic acid encoding a non-naturally occurring protein comprising a native flexibly linked trimer (NFL), wherein the NFL comprises an HIV-1 envelope glycoprotein (Env) linked to a natural HIV transmembrane domain (HIV TM), and wherein the HIV TM is further linked to a cytoplasmic tail (CT).

[0028] In some embodiments, the nucleic acid is formulated in a nanoparticle.

[0029] In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0030] According to another aspect of the present disclosure, the present disclosure provides for method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of a non-naturally occurring protein comprising a native flexibly linked trimer (NFL), wherein the NFL comprises an HIV-1 envelope glycoprotein (Env) linked to a natural HIV transmembrane domain (HIV TM), and wherein the HIV TM is further linked to a cytoplasmic tail (CT).

[0031] According to another aspect, the disclosure provides an immunogenic composition comprising any of the proteins or nucleic acids described herein and a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises an adjuvant.

[0032] According to another aspect of the present disclosure, the present disclosure provides for a method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the nucleic acid encoding a non-naturally occurring protein comprising a native flexibly linked trimer (NFL), wherein the NFL comprises an HIV-1 envelope glycoprotein (Env) linked to a natural HIV transmembrane domain (HIV FM), and wherein the HIV TM is further linked to a cytoplasmic tail (CT).

[0033] According to another aspect of the present disclosure, the present disclosure provides for a non-naturally occurring mRNA encoding a non-naturally occurring protein comprising a native flexibly linked trimer (NFL), wherein the NFL comprises an HIV-1 envelope glycoprotein (Env) linked to a natural HIV transmembrane domain (HIV TM), and wherein the HIV TM is further linked to a cytoplasmic tail (CT).

[0034] In some embodiments, the mRNA is formulated in a nanoparticle.

[0035] In some embodiments, the nanoparticle a lipid nanoparticle (LNP).

[0036] According to another aspect of the present disclosure, the present disclosure provides for a method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA encoding a non-naturally occurring protein comprising a native flexibly linked trimer (NFL), wherein the NFL comprises an HIV-1 envelope glycoprotein (Env) linked to a natural HIV transmembrane domain (HIV TM), and wherein the HIV TM is further linked to a cytoplasmic tail (CT).

[0037] In some embodiments, the animal is a mammal.

[0038] In some embodiments, wherein the mammal is a human.

[0039] In one aspect, the present disclosure provides for a protein of any one of claims 1-21 for eliciting an immune response by a method comprising systemically administering to an animal in need thereof an effective amount of the protein.

[0040] In some embodiments, the animal in need thereof is a mammal.

[0041] In some embodiments, the mammal is a human.

[0042] In another aspect, the present disclosure provides for a nucleic acid of any one of claims 22-24 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the nucleic acid.

[0043] In some embodiments, the animal in need thereof is a mammal.

[0044] In some embodiments, the mammal is a human.

[0045] In yet another aspect, the present disclosure provides for an mRNA of any one of claims 31-33 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA.

[0046] In some embodiments, the animal in need thereof is a mammal.

[0047] In some embodiments, the mammal is a human.

[0048] In one aspect of use, the present disclosure relates to the use of a protein of any one of claims 1-21 for eliciting an immune response by a method comprising systemically administering to an animal in need thereof an effective amount of the protein.

[0049] In some embodiments of this use, the animal is a mammal.

[0050] In some embodiments of this use, the mammal is a human.

[0051] In another aspect of use, the present disclosure relates to the use of an mRNA of any one of claims 31-33 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA.

[0052] In some embodiments of this use, the animal is a mammal.

[0053] In some embodiments of this use, the mammal is a human.

[0054] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112(a)) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior fded application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0055] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0056] These and other embodiments are disclosed or are obvious from and encompassed by the following Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The patent or application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0058] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0059] Figure 1. Design of the cell surface single chain HIV Env mimic NFL.711. (A, top) Schematic of the HIV Env gpl60 precursor displaying the surface subunit gpl20 (blue) and the membrane subunit gp41 ectodomain (brown), the transmembrane domain (TM, green) and the cytoplasmic tail (purple). The natural HIV Env cleavage site (sosREKRsii) between gpl20 and gp41 is shown. (A, bottom) Schematic of the soluble Env NFL.664 construct. The heterologous CD5 leader (pink) and the engineered linker (2xG4S) are indicated. Not shown in the diagram for simplicity but included in the stabilized NFL design are a cassette of stabilizing mutations designated as “TD CC+” (See Figure 9). (B) Schematic of the membrane-bound NFL.711 constructs without (- MPER, top) and with (+MPER, bottom) the membrane proximal external region (MPER). The NFL.711 construct follows a similar stabilization design as the soluble NFL.664 trimer (TD CC+, see Figure 9). The (-MPER) design includes a second short G4S linker in place of the MPER. (C) Reconstruction of the membrane-bound HIV Env showing the cryo-EM tomography density of the natural HIV Env (gray) with an underlying colored ribbon crystal structure that indicates the different regions of Env. The following structures and EM densities were used to make this reconstruction (16055 NFL TD CC+ PDB ID 5UM8, MPER and TM regions PDB ID 6E8W, CT region PDB ID 7LOH and for the EM Spike density EMD-5272)

[0060] Figure 2. Antigenicity by flow cytometry on cells transfected with plasmid DNA encoding NFL.711 constructs. (A) Test case membrane-bound 1086cNFL.711 trimers expressed from plasmid DNA. Selected antibodies were used (25 pg / mL starting concentration, serially dilute 5-fold) to determine binding to trimeric NFLs expressed on the cell surface of HEK293 T cells by flow cytometry. The inventors compare the (-MPER) construct, lacking the membrane proximal external region, (left) with the (+MPER) (right) design on the 1086c Env. The datapoints represent the average MFI levels and SEM resulting from two independent experiments expressing NFL.711 proteins. Transfected cells were stained with the broadly neutralizing antibodies bnAbs (BG18, PGT145, VRC01, 10E8 and 2F5, the latter not neutralizing for the 1086c HIV strain) and the non- broadly neutralizing antibodies (Fl 05 and 447-52D). The relative binding of these antibodies alludes to the conformational state of the trimeric proteins on the cell surface. (B) Similarly, as above, the inventors tested the antigenicity of three additional NFL.711 constructs (+MPER) derived from a 655 clade B, JR-FL, a clade A, BG505 and a clade C, 16055 Env sequences that extend the applicability of the cell surface design.

[0061] Figure 3. Antigenicity by flow cytometry of NFL.711 proteins expressed from mRNA LNPs. Bars represent MIF values with SEM corresponding to the binding of an antibody to a trimeric membrane-bound Env protein expressed on the cell surface of the HEK293 T cells transfected with mRNA LNPs encoding the NFL.711 (+MPER) constructs. Here, it is shown the MFI values as bars with SEM corresponding to two separate experiments at two concentrations of the antibodies, 25 pg / ml (Top) and 2.5 pg / ml (bottom).

[0062] Figure 4. Immunogenicity schedule and immunogen regimens. Six groups of five rabbits were immunized 4 times following the immunogen regimens and the schedule shown. Colored in light blue are the groups of animals immunized with mRNA LNPs and colored in light orange the two groups that received soluble protein in adjuvant. The animals were immunized four times at 0, 4, 12 and 24 weeks as denoted with white arrows. Bleeds were collected at the time points specified in the figure with red arrows.

[0063] Figure 5. ELISA serum binding titers at the two-week timepoint following each immunization. (A) Box and whisker plot corresponding to serum binding titers of animals that received mRNA LNPs inoculations displaying individual area under the curve (AUC) values, minimum and maximum values, and the group median. Each color represents a different Env immunogen, orange for 1086c NFL.711, green for BG5O5 NFL.711, blue for JR-FL NFL.711 and pink for 16055 NFL.711 (B) Comparison of ELISA serum binding titers of animals that received either mRNA LNP inoculations or a HIV Env matched soluble protein control. Left graph shows data corresponding to animals immunized with 1086c NFL.711 encoding mRNA LNPs in orange or soluble protein in red. Likewise, on the right, ELISA titers are shown corresponding to animals that received JR-FL NFL.711 encoding mRNA LNPs in light blue or matched soluble protein in dark blue.

[0064] Figure 6. HIV-1 neutralization titers of autologous tier-2 virus, (left) Serum dilution ID50s are shown for three consecutive time points corresponding to bleeds collected after the second, third and fourth inoculations. (Right) Purified total IgG inhibitory concentrations, IC50s are shown after the fourth inoculation for comparison and confirmation of inhibition of entry mediated by immunoglobulin. The values shown represent the serum dilution (left) or the concentration (pg / mL) of total IgG (right) necessary to inhibit 50% of viral entry in a HIV-1 pseudovirus TZM-bL assay. An arbitrary colorimetric scale was used to highlight the variation in neutralization potency of both the serum and IgG samples.

[0065] Figure 7. Electron microscopy polyclonal epitope mapping (EMPEM) of selected animal sera. Blue rectangles (left) show the EM densities of the corresponding NFL trimer in complex with the polyFabs from the two animals immunized with mRNA LNPs while orange rectangles (right) show the densities of the animals immunized with the Env (top 1086c and bottom JR-FL) matched soluble proteins. The trimer densities are colored in gray while the polyFabs are colored green, blue, yellow, and purple.

[0066] Figure 8. Glycan profiles of the soluble and membrane-bound NFL trimers. Each horizontal bar displays the proportion of unoccupied (gray), complex (magenta) or high-mannose (green) N-glycans at the Env position numbered to the left of the bar. Significant changes in glycan content are indicated by color coordinated arrows. Pie charts display the average content of unoccupied, complex and high- mannose percentage detected in the two variants of the trimeric NFL proteins, soluble (left) and membrane-bound (right).

[0067] Figure 9. NFL trimer stabilizing mutations (TD CC+). All four HIV Env sequences utilized here to make either membrane-bound or soluble 'NFL proteins were modified as shown in the figure. Each mutation is categorized as followed: TD mutation refers to BG505 Trimer Derived mutation (Guenaga et al, J Virol 2015); V3 refers to V3 loop stabilizing mutations (Guenaga et al, Immunity 2017); FP refers to Fusion Peptide stabilizing mutation (Guenaga et al, Immunity 2017); Helix disrupting refers to proline or glycine residues in gp41 (Sanders et al, I Virol 2002, Guenaga et al, Immunity 2017); Disulfide refers to an introduce Cys-Cys covalent linkage introduced to prevent CD4 conformational changes (Kwon et al, Nat Struct Mol Biol, 2015, Guenaga et al, J. Virol 2015).

[0068] Figure 10. SDS-PAGE analysis of the immunoprecipitated NFL.711 proteins under reducing conditions. A panel of 4 antibodies (F105, VRC01, PGT145 and 10E8) were used to selectively purify by immunoprecipitation the membrane bound trimeric Env expressed on the cell surface of HEK 293T cells transfected with the NFL.711 constructs after solubilization with detergent Triton X-100. The different intensity of the Env bands alludes to the selective expression levels of each construct and the affinities of that specific Env for the antibodies tested. Top gel shows a direct comparison of the same 1086c NFL with and without the MPER region. The bottom gel shows the other Env derived NFL.711 (+MPER) 16055, BG505 and JR-FL.

[0069] Figure 11. FACS binding of selected antibodies to NFL.711 membrane-bound proteins. The V3-N332 glycan targeting antibody 2G12, the MPER targeting 4E10 and the Fusion Peptide targeting VRC34 were used to assessed binding to JR-FL and 1086c NFLs expressed on the cell surface after DNA plasmid transfection of HEK293 T cells.

[0070] Figure 12. Dose dependent expression of the BG505 NFL.711 expressed from mRNA LNPs. (A) mRNA dose effect on cell-surface expression of the BG505 NFL.711 proteins in transfected HEK 293T cells as assessed by antibodies 10E8 (blue) and F105 (black) with a secondary only negative control. (B) Comparison of cell surface and intracellular expression of BG505 NFL proteins after transfection of HEK293T cells with increasing amounts of mRNA. (C) Relative amounts of cell surface and intracellular proteins after 5 pg mRNA transfections of BG505, 16055 and JR-FL measured by mAb binding 10E8.

[0071] Figure 13. Correlation between binding and neutralization. The top graph displays the non-significant correlation between ELISA serum binding titers and serum neutralization(ID50s) while the bottom graph shows the, also non-significant, correlation between FACS binding of serum IgG titers and the corresponding serum IgG IC50s of neutralization.

[0072] Figure 14. Correlation between binding and neutralization. Animal sera (left) and purified IgG samples (right) were tested in a TZM-bl assay against a panel of tier 1 HIV-1 pseudoviruses (SF162, HXB2 and MN) and VSV as a negative control. Values in the table signify the reciprocal serum dilution (ID50s, left) and purified IgG inhibitory concentrations (IC50s, right) at which 50% of the virus entry is inhibited. An arbitrary colorimetric scale was used to highlight the range of serum and IgG potency of inhibition of viral entry.DETAILED DESCRIPTION OF THE INVENTION

[0073] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0074] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like, reference to “a B cell” includes a plurality of such B cells, and reference to “the antibody” includes reference to one or more antibodies and equivalents thereof known to those skilled in the art.

[0075] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a valueis disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as values between 10 and 15. For example, it is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In this application, if a data point range is disclosed, it is understood that each unit from the lowest data point to the highest stated datapoint, including the first (lowest) and last (highest) data point is disclosed. For example, if a data point range 1-20 is disclosed, it is understood that data points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and each unit between any two particular units in the range are also disclosed. It is also understood that whenever a series of values are disclosed, that any range falling between any two of the recited values is also understood to be included.

[0076] The terms “pharmaceutical composition” “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes embodiments where said event or circumstance occurs and embodiments where it does not.

[0077] The term “isolated” or “non-naturally occurring” is used herein to indicate that the isolated moiety (e.g. peptide or compound) exists in a physical milieu distinct from that in which it occurs in nature. For example, the isolated peptide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs. The absolute level of purity is not critical, and those skilled in the art may readily determine appropriate levels of purity according to the use to which the peptide is to be put. In the context of the present invention, this refers to the NFL trimer proteins or nucleic acids encoding them, which have been designed and produced by recombinant means and are distinct from any viral Env protein found in nature.

[0078] The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence” are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and itmay be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component. The immunogenic trimers of the present disclosure are polypeptides.

[0079] As used herein, the terms “antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and / or cellular type directed against that protein. An antigen can be a protein, a polypeptide, a peptide, a polysaccharide, a lipid, or a nucleic acid, or a combination thereof. In an aspect, an antigen of the present disclosure is a membrane-bound HIV-1 Env NFL trimer.

[0080] The term “antibody” includes intact molecules as well as fragments thereof, such as Fab, F(ab’)2, Fv and scFv which are capable of binding the epitope determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor. An “isolated antibody“ or “non-naturally occurring antibody“ is one that has been separated and / or recovered from a component of its natural environment. The term “monoclonal antibody“ as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies which may comprise the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0081] A “neutralizing antibody” is an antibody that reduces or abolishes the infectivity of a pathogen, for example by inhibiting the entry of HIV-1 virus. Broad and potent neutralizing antibodies may neutralize greater than about 50% of HIV-1 viruses (from diverse clades and different strains within a clade) in a neutralization assay. The immunogens of the present disclosure are designed to elicit such neutralizing antibodies.

[0082] As used herein the terms “nucleotide sequences” and “nucleic acid sequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. The nucleic acid may be single-stranded, or partially or completely double-stranded (duplex). Duplex nucleic acids maybe homoduplex or heteroduplex. This disclosure particularly relates to mRNA sequences encoding the immunogenic trimers described herein.

[0083] As used herein the term “transgene” may be used to refer to “recombinant” nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present disclosure. The term “recombinant” means a nucleotide sequence that has been manipulated “by man” and which does not occur in nature, or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man” means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc. The nucleic acids encoding the NFL trimers of the disclosure are recombinant.

[0084] As used herein, “codon optimization” refers to the modification of a nucleic acid sequence for improved expression in a particular host cell or organism, without altering the amino acid sequence of the encoded polypeptide. This can involve replacing codons that are rarely used by the host with codons that are more frequently used, to enhance translational efficiency. The nucleic acids of the present disclosure, particularly the mRNAs, are advantageously codon- optimized for expression in mammalian, and specifically human, cells.

[0085] For the purposes of the present disclosure, sequence “identity” or “homology” is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms such as the algorithm of Karlin & Altschul (1990, 1993), Myers & Miller (1988), or the FASTA algorithm (Pearson & Lipman, 1988).

[0086] As used herein, an “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. For the purposes of this disclosure, this can refer to plasmid DNA vectors used for in vitro characterization and mRNA production.

[0087] The term “pharmaceutical composition” is used herein to define a solid, liquid, or particulate composition in a form, concentration, and level of purity suitable for administration to a subject (e g., a human patient). The terms “immunogenic composition,” “immunological composition,” and “vaccine composition” or “vaccine” cover any composition that elicits an immune response against the targeted pathogen, HIV, and advantageously induces a protective immune response.

[0088] “Adjuvants” are any substance whose admixture with an administered antigen increases or otherwise modifies the immune response to said antigen. Adjuvants may for example be selected from the group consisting of A1K(SO4)2, AlNa(SO4)2, A1NH4(SO4), silica, alum, Al(0H)3, oil-in-water emulsions such as MF59, and Toll-like receptor (TLR) agonists.

[0089] “Subject” refers to mammals and includes human and non-human animals. In a preferred aspect, the subject is a human at risk for or infected with HIV.

[0090] A “conservative amino acid change” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid and glutamic acid), non-charged amino acids or polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), beta-branched side chains (e.g. threonine, valine and isoleucine), and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan and histidine). The immunogenic trimers of the disclosure may contain conservative amino acid substitutions provided they retain their desired antigenic properties.

[0091] As used herein, “administering” or “administration” refers to the act of giving a drug, prodrug, or other agent, or a vaccine or immunogenic composition, to a subject. For the present disclosure, this typically refers to parenteral routes, such as intramuscular injection.

[0092] An “effective amount” or “therapeutically effective amount” of an agent (e.g., an mRNA-LNP vaccine) refers to an amount sufficient to elicit a desired biological or medical response, such as an immune response, or to alleviate, ameliorate, prevent, or delay the onset or progression of a disease or condition, such as HIV infection or AIDS.

[0093] The term “lipid nanoparticle” or “LNP” refers to a particle comprising one or more lipids, such as a cationic or ionizable lipid, a non-cationic lipid, a sterol, and a PEG-lipid, which encapsulates a nucleic acid, such as an mRNA molecule encoding an NFL trimer of the disclosure.

[0094] As used herein, the terms “eliciting an immune response,” “immunogenic response,” or “vaccine response” refer to the development in a subject of a humoral and / or a cell-mediated immune response to a composition or vaccine of interest. For example, such a response can include the production of antibodies, including neutralizing antibodies, and / or the activation or proliferation of T cells specific for an antigen present in the composition or vaccine.

[0095] As used herein, the term “flexible linker” refers to an engineered peptide sequence designed to connect distinct protein domains, replacing natural cleavage sites or bridging gaps between domains. Such linkers, like (G4S)2 or G4S, impart flexibility and facilitate the proper folding and function of the resulting chimeric protein.

[0096] Previously, it was demonstrated that furin cleavage of full-length native Env is required for membrane bound spikes to display a favorable antigenic profile, consistent with the pre-fusion conformation of native Env and consistent with the similar requirement needed to generate well- ordered SOSIP trimers (22, 23). Overcoming the requirement of furin cleavage would be preferable for genetic expression of Env trimers, eliminating the need to co-express exogenous furin to ensure complete cleavage, a prerequisite for a well-ordered natural HIV Env or SOSIP conformation. For this reason, the NFL design and subsequent stabilized developments should be ideal for genetic expression where it is not possible to selectively purify well-ordered trimers by negative or positive selection (7, 9, 10, 14, 16, 24).

[0097] Here, the stabilized NFL design is extended to generate membrane-bound NFL trimers following genetic expression from both DNA and mRNA. For this, follow two design strategies were followed: either the NFL.664 trimer was tethered to the HIV TM via a second short flexible linker. Or, alternatively, the full MPER region was incorporated into the soluble trimer design, restoring the full Env ectodomain, and followed by the natural HIV TM and cytoplasmic tail (CT). In both soluble SOSIP and NFL trimers, the MPER was removed to avoid trimer aggregation and misfolding (25, 26). In a second design, it was reasoned that it might be possible to restore this conserved, broadly neutralizing determinant in the membrane context without detriment to the native-like properties of these novel trimers expressed on the cell surface, which it was demonstrated in vitro and in vivo by elicitation of autologous tier-2 neutralizing serum antibody responses in immunized rabbits with three HIV strains. Accordingly, the foregoing demonstrates developments for genetic expression of native-like Env trimers in animals and in humans as vaccine candidates.

[0098] Design of membrane-bound HIV Env NFL trimers for genetic expression

[0099] To design cell-surface membrane-bound NFL trimers, that from hereon are referred to as NFL.711 (number refers to the last residue of HIV Env incorporated in the construct), the inventors introduced several modifications from the natural Env sequence (see Figure 1A and Figure 9). To begin, the inventors used a heterologous CD5 leader sequence in place of the naturalHIV signal sequence to increase Env trimer expression (34). Next, the inventors codon optimized the natural HIV Env sequences to eliminate the RRE regulatory stem loop and render Env mRNA export to the ribosomes independent of rev co-expression. In addition, the inventors modified natural Env coding for residues that incorporate the cassette of stabilizing mutations developed to generate homogeneous soluble HIV Env NFL trimers (see Figure 1A and Figure 9 for Env mutation details). It was reasoned that this successful stabilizing strategy for the soluble NFL.664 trimers would transfer well to membrane bound, cell-surface NFL trimer expression (9, 10, 35- 37). As a test case, the inventors selected the African HIV- 1 1086c viral Env sequence to design membrane-bound NFL trimers because previously this Env strain was successfully used to improve the proportion of well-ordered soluble trimeric Env proteins expressed following transient expression (10). Fhe inventors retained features of the original NFL design that include a flexible linker (G4S)2 between the gp!20 and gp41 subunits replacing the natural furin cleavage site, REKR, at the C-terminus of gpl20, thus, rendering the trimer cleavage-independent (Figure 1A)(7). The inventors used two different strategies to link the Env ectodomain to the TM. In the first design, the inventors employed a second small G4S linker to ‘fill the gap’ between the C- terminus of the NFL Env at residue 664 and the natural HIV TM beginning at residue 684. This linker bridging Env residues 664 to residue 684 (original HXBc2 numbering) facilitates the linkage to the TM in the absence of the natural MPER (Figure IB, top). The second strategy restored the full natural MPER region (665-683) followed by the natural TM (Figure IB, bottom). Finally, to increase cell-surface expression, the inventors truncated the natural long Env CT at residue 711, removing the normal endocytosis signals that can limit Env on the plasma membrane (38, 39) (Figure IB). These modifications should result in trimers embedded in the plasma membrane from cells transiently transfected with genetic material both in vitro and in vivo (Figure 1C).

[0100] Expression and antigenicity of membrane-bound NFL Envs on the cell surface following DNA transient transfection is consistent with a near-native conformation

[0101] Initially, the inventors sought to determine if the newly designed 1086c NFL.711 construct generated membrane-bound trimeric Env on the cell surface of transiently transfected HEK 293T cells. By flow cytometry, the inventors assessed cell-surface Env expression and conformational state of the expressed proteins by ‘antigenic profiling’. Selected HIV antibodies were used for this analysis: PGT145 and VRC26, broadly neutralizing antibodies (bnAb) that exclusively recognize a quaternary epitope of native Env(40-42); VRC01, a CD4 binding-sitebnAb (43); BG18, a bnAb that targets the N332 glycan supersite; and two MPER-directed bnAbs, 10E8 and 2F5 (44, 45), to validate that their respective target sites are present and accessible in the trimeric membrane-bound proteins. In addition, the inventors selected two non-neutralizing antibodies (unable to neutralize ‘tier-2’ clinical isolates), Fl 05 (CD4bs-directed) (46) and 447- 52D (V3-directed)(47), that recognize their epitopes on 382 undesirable open forms of Env and cannot access their epitopes on well-ordered and well-folded trimeric spikes. A desirable antigenic outcome would retain binding of the bnAbs while minimizing the binding of F l 05 and 447-52D antibodies, indicating that the expressed trimeric proteins on the cell membrane adopt a well- ordered native-like conformation. Following transient expression from plasmid DNA encoding 1086c NFL.711 constructs, the inventors analyzed both 1086c variants, lacking or incorporating the MPER, named “-MPER” (lacking the MPER) and “+MPER” (possessing the natural MPER). It was determined that both trimer types were expressed on the cell surface of transfected HEK293 T cells with the (+MPER) variant having higher levels of expression as assessed both by flow cytometry and by gel electrophoresis after antibody immunoprecipitation (Figure 2A and Figure 10). Both constructs were efficiently recognized by the bNAbs PGT145, VRC26, BG18 and VRC01 and minimally by the non-neutralizing Fl 05 and 447-52D mAbs, Their antigenic profiles indicated that the majority of the expressed trimers expressed on the cell surface retained a nativelike conformation (Figure 2 A). Consistent with design criteria, the MPER-directed bNAb, 10E8, recognized only the +MPER construct but not the -MPER trimer construct because the domain was genetically removed in the latter design (Figure 2A, right and Figure 9). In agreement with its inability to neutralize clade C strains, the MPER-directed but clade B-restricted 2F5 bNAb did not bind the +MPER clade C 1086c NFL.711 trimers. Based on these encouraging results, the inventors expanded the cell surface design NFL.711 (+MPER) to three other Env strain sequences, namely, BG5O5, JR-FL and 16055 (Clades A, B and C, respectively) to determine more general applicability. The new three constructs followed the same general design as done for 1086c (+MPER) since this design generated higher expression of surface Env and contained additional bNAb target sites. Following production of plasmid DNA and transient transfection, the inventors then assessed expression by flow cytometry and determined the level of binding by the same set of antibodies. This next set of membrane-bound trimers were well recognized by the bNAbs PGT145, BG18, VRC01 and by the MPER-directed 10E8 but minimally recognized by the nonneutralizing antibodies F105 and 447-52D (Figure 2B). The 2F5 antibody selectively recognizedmembrane-bound NFL trimers corresponding to Envs from HIV strains for which it neutralizes: BG505 and JR-FL. The antigenic profding suggested that the membrane-bound trimeric proteins retained a predominantly native-like trimer conformation.[001021 In parallel, the inventors expressed the membrane-bound proteins in HEK 293T cells and extracted the cell surface Env NFL.711 proteins with a 0.5% triton solution to solubilize the membrane-bound trimers. A panel of four antibodies, PGT145, 10E8, VRC01 and Fl 05 was used to immuno-precipitate the Env proteins and performed polyacrylamide SDS-gel electrophoresis to assess both conformation and relative level of expression (Figure 10). This analysis was consistent with that of the flow cytometry, indicating that all four Env strains were largely expressed in a native-like conformation recognized by the quaternary antibody PGT145 (Figure 10).

[0103] Cell-surface NFLs are well-expressed from mRNA lipid nanoparticles in vitro and retain a native-like conformation

[0104] To determine in vitro expression of the four Env NFL.711 trimers from mRNA the inventors generated 1086c, BG505, IR-FL and 16055 NFL.711 (+MPER) encoding, nucleoside- modified mRNA encapsulated lipid nanoparticles. These constructs are identical at the amino acid level to those encoded by the DNA plasmids described in the previous section, however, the coding nucleotide sequences were codon optimized appropriately to enhance expression from mRNA. Following transfection of HEK 293T cells with the mRNA lipid nanoparticles, the inventors assessed Env trimer expression by FACS and antigenicity of the membrane-bound Env proteins. Following a similar approach to the antigenic characterization of the constructs expressed from plasmid DNA, the inventors assessed binding of antibodies PGT145, VRC01, 10E8, F105 and 447-52D (40, 42, 43, 46, 48, 49) to determine the antigenic properties of the Env proteins expressed from mRNA. Two antibody concentrations, 25 pg and 2.5 pg per ml for FACS analysis were used in two independent experiments. The data was plotted as bars representing the mean fluorescence intensity (MFI) with SEM corresponding to each antibody binding to a membrane-bound NFL trimer (Figure 3). The 1086c, IR-FL and 16055 NFL.711 proteins expressed robustly from mRNA LNPs based on PGT145, VRC01 and 10E8 bNAb recognition at both antibody concentrations 25 pg / mL and 2.5 pg / mL (Figure 3). Recognition by the non-neutralizing Fl 05 and 447-52D antibodies was not distinguishable from controls (‘cells only’ negative controls), indicating that the trimeric proteins expressed on the cell surface from mRNA were well-ordered native-like Env trimers, in alignment with observations from DNA plasmid transfections. In contrast, expressionof BG505 NFL.711 trimers from mRNA LNPs was barely detectable at the highest antibody concentration tested (Figure 3, top). While the expression and binding results for the 1086c, 16055 and JR-FL constructs were comparable to those acquired from DNA plasmid transfections, the BG505 expression markedly differed (Figure 3 and 2B). Next, it was investigated if a higher dose of the mRNA would increase BG505 NFL.711 expression. Accordingly, a dose titration experiment was performed where in which the inventors transfected HEK 293T cells with increasing amount of mRNA 5, 10, 30 and 40 pg of mRNA LNPs encoding BG505 NFL.711. The inventors then assessed BG505 NFL.711 trimer expression by FACS using 10E8 and F105 as the detecting antibodies, as 10E8 binds to a linear epitope on trimers and Fl 05 will bind to open forms of Env, assuring us of detecting all forms of Env. Higher BG505 NFL.711 Env expression was detected with increasing amounts of mRNA as determined by increasing levels of 10E8 binding, however still relatively low expression even at 8 times the amount of mRNA transfected (Figure 11 A). BG505 NFL.711 expression was considerably lower than that of 1086c, JR-FL and 16055 NFL.711 based on the level of mean fluorescence intensity detected (Figure 3). Following cell permeabilization using the Cytofix / Cytoperm Fixation / Permeabilization kit, (BD Cat. No. 554714), it was determined by intracellular flow cytometry antibody staining that much of BG505 Env was disproportionally retained in the cytoplasm while only a fraction was transported to the cell surface (Figure 1 IB). For other Envs tested in this manner, the relative amount of 16055 and JR-FL Env trimers detected on the cell surface was larger than that of BG505 (Figure 11C).

[0105] In sum, 1086c, JR-FL and 16055 NFL.711 membrane-bound trimers were well- expressed and generated predominantly well-ordered trimeric proteins on HEK293 T cells transfected with mRNA LNPs while BG505 Env was poorly expressed on the cell-surface.

[0106] Membrane-bound NFL trimers are immunogenic following multiple inoculations with mRNA LNPs in vivo

[0107] The inventors next conducted a rabbit immunogenicity study testing mRNA LNPs encoding the trimeric membrane-bound NFL.711 proteins expressed in vivo. Four groups of five rabbits each were inoculated with 1086c, BG505, JR-FL and 16055 NFL.711 encoding mRNA LNPs four times at 0, 4, 12 and 24 weeks with 5 pg of mRNA via the intramuscular route (Figure 4). To compare mRNA to soluble protein delivery the inventors added two additional groups that received 25 pg of 1086c and JR-FL stabilized (TD CC+, see Figure 9) NFL.664 soluble proteins in adjuvant, the standard protein dose for small animals (Figure 4). ELISA binding revealed thatall immunogens generated antigen-specific serum antibody binding responses after four inoculations (Figure 5). Antibody binding responses varied in magnitude between the different mRNA immunogens and amongst animals. The group that received the mRNA LNPs encoding BG505 showed the lowest antibody responses. In contrast, groups that received mRNA LNPs encoding 1086c, JR-FL and 16055 showed higher serum binding antibody responses at all time points compared to BG505 (Figure 5A). When comparing soluble protein to mRNA LNP immunization, the animals that received 474 soluble protein in adjuvant showed slightly higher and accelerated serum antibody binding responses compared to their HIV-strain-matched mRNA groups (Figure 5B). Serum titers of the animals inoculated via mRNA LNPs did not reach a saturation point even after 4 inoculations, suggesting that perhaps they may have benefited from additional inoculations or a higher dose (Figure 5A).

[0108] Additionally, the inventors tested the serum for inhibition of HIV entry in a TZM-bl neutralization pseudovirus assay (see Methods). This assay was performed in two different ways: with serum samples (post 2 week 6, post 3 week 14, and post 4 week 26) serially diluted from a starting dilution of 1 : 10 and then confirmed with protein-A-purified serum IgG (post 4 week 26) starting at 2000 pg / mL of total IgG (Figure 6). The total IgG starting dilution is based in the average content of IgG in serum, approximately 10-15 mg / mL. This starting IgG concentration (2000 pg / mL) is approximately equivalent to a 1 :5 serum dilution in its capacity to inhibit HIV entry, so about a 2-fold gain in potency and detectability with respect to the serum assays. A low dilution of the sera is equivalent in potency to a higher concentration of the purified total IgG, essentially the ID50s and IC50s are inverted in value (Figure 6). Taking both neutralization formats into account, autologous tier-2 virus neutralization was detected in three out of four groups of animals receiving mRNA except for the low binding titer BG505 group of animals (Figure 6). The autologous neutralization activity was detected after 2 or 3 inoculations in selected animals and improved with subsequent immunizations (Figure 6, left). The strongest neutralizing titers were observed in the 1086c group where all five animals generated strong autologous tier-2 neutralizing titers (Figure 6). Likewise, all five animals immunized with JR-FL NFL.711 mRNA generated autologous tier-2 neutralization antibody responses that varied in magnitude as detected in the purified IgG assay format (Figure 6, right). Three out of five animals immunized with 16055 NFL.711 mRNA generated robust autologous responses in agreement with past observations in rabbits utilizing stabilized 16055 NFL.664 soluble trimers as immunogens (Figure 6)(35).

[0109] When comparing autologous neutralizing antibody titers between groups of animals that received mRNA LNPs to those that received strain-matched adjuvanted soluble protein immunizations, the number of responder animals developing autologous tier-2 1086c and JR-FL neutralization titers was the same after 4 immunizations (Figure 6). However, the animals that received JR-FL soluble-protein generated slightly stronger autologous neutralization titers and binding titers (Figure 6 and 5, respectively). Neutralizing antibody titers against highly sensitive Tier-1 viruses (MN.03, SF162 and HXBc2) were more robust in the soluble protein groups than in the matched mRNA groups (Figure 12). These results suggest that the V3 loop in the trimers, main target of these tier-1 neutralizing serum antibodies, may be less exposed in membrane- anchored NFL trimers than in the soluble counterparts (Figure 12). These results agree with observations made in a recently published study comparing CH505 HIV Env immunogens delivered via mRNA LNP to soluble SOSIP trimeric proteins (19).

[0110] In sum, it was concluded that 1086c, JR-FL and 16055 NFL.711 constructs delivered by mRNA LNPS are immunogenic and generate an autologous tier-2 neutralizing antibody response against the matched HIV-1 strain and equivalent to their Env matched soluble protein immunogens. These neutralization data represent strong evidence that the membrane-bound stabilized NFL.711 trimers expressed from mRNA in vivo are close mimics of the native spike.

[0111] EMPEM analysis of serum antibody responses reveals sites of vulnerability in 1086c and JR-FL HIV-1 strains

[0112] To understand in more detail the vaccine-elicited serum antibody responses by both mRNA and soluble protein immunogens, the inventors selected four animals displaying robust and comparable autologous neutralizing antibody titers representing the two classes of immunogens, mRNA LNPs and soluble protein (animal ID numbers: U5919 and U5403 from the 1086c-based immunizations; and U5756 and U5902 from the JR-FL; respectively, see Figure 6 and 7) for electron microscopy polyclonal epitope mapping (EMPEM) analysis (50, 51). EMPEM allows us to identify Env epitopes targeted in the vaccine-elicited immune response circulating in the serum IgG. For this analysis, polyclonal IgG was digested to Fabs (polyFab) from the serum samples collected after four immunizations (Post 4 week 26) and combined with the immunogen matched soluble trimer, either 1086c NFL.664 or JR-FL NFL.664. EMPEM revealed several specificities targeting both the 1086c and JR-FL envelope glycoproteins as detected by the polyFab densities bound to trimeric Env (Figure 7). For the 1086c Env, polyFab generated by mRNA LNPinoculation displayed three binding targets, two in the gpl20 domain (V2V3 region and C3 regions) and a gp41 trimer-base-directed response (Figure 7, top left). In comparison, the polyFabs generated in the rabbit immunized with soluble 1086c trimeric proteins displayed the same C3 and trimer base specificities but, in addition, a distinct gp41 interface specificity (Figure 7, top right). The C3-directed polyfab density present in both animals overlaps with a natural absence of glycosylation sites at Env positions 356 and 360 in the natural 1086c HIV envelope sequence creating a hole in the glycan shield. Antibodies targeting this glycan hole are likely one of the specificities that mediates the neutralization activity against the 1086c pseudovirus since it shows in the EMPEM analysis as a polyFab (blue) in the samples of the two animals with highest neutralizing antibody titers (Figures 6 and 7). Glycan holes resulting from N-glycans absent at specific sites are the main neutralization determinants for other strains of HIV (52, 53). It was noted that the mRNA sample showed a V2V3 targeting antibody response, suggesting that membranebound trimer array may be beneficial to generate Env-apex targeting serum antibodies as previously demonstrated in a knock-in mouse model (54).

[0113] For the JR-FL Env, the mRNA samples revealed two specificities being targeted, the C3 / V5 region in gpI20 and a base-directed gp41 response (Figure 7, bottom left). In contrast, the soluble protein sample showed three specificities, a gpl20 interface, a gp41 interface and a trimer base-directed response. The C3 / V5 region of gpI20, located adjacent to the CD4bs has been identified as a neutralization determinant for other strains of HIV (55). In JR-FL Env this region is missing a glycosylation site at N460 which might explain the elicitation of antibodies targeting the V5. Similarly, the gpI20 interface in the JR-FL Env is missing a glycosylation site at position 197 creating a glycan hole that may allow access to a generally well-occluded epitope. The only polyFab density found in both samples was to the trimer-base which generally corresponds to not neutralizing or weakly neutralizing antibody responses. More likely, the EM densities of PolyFabs targeting the C3 / V5 and the gpl20 interface correspond to antibodies mediating autologous neutralization of HIV-1 JR-FL. Both Env sites lack N-glycans at residues N460 and N197, respectively.

[0114] To note, the EMPEM data derived from the animals that were immunized with soluble protein showed unique densities targeting the gp41 interface (yellow polyFab density, Figure 7, right), which are absent from the samples derived from animals that received mRNA LNP vaccinations (Figure 7, left). This gp41 density is associated with a non-neutralizing antibodyresponse towards a gp41 region generally occluded by N-glycans at residues 611, 616 and 637. It is viewed as an undesirable consequence of vaccination with soluble Env proteins displaying gp41 N-glycan sites not fully occupied (55-57). Regarding this, the inventors analyzed the glycan profdes of the soluble and membrane-bound 16055 NFL trimers by mass spectrometry (58). This analysis revealed significant differences in the composition and occupancy of N-glycosylation sites between the soluble and membrane-bound NFL trimers (Figure 8). It was observed that statistically significant increases in N-glycan occupancy at the 611 N-glycan site in the membranebound trimer compared to the soluble trimer that might explain the absence of the gp41 targeting polyFabs in the samples derived from the mRNA vaccinations. Moreover, the gp41 N-glycan sites presented significant increases in the proportion of high-mannose N-glycans and decreases in complex N-glycans in the membrane-bound trimer suggesting that the anchorage in the membrane or the rigidity of the quaternary structure of these stabilized trimers might sterically hinder access of glycan processing enzymes to the base of the trimer. The high-density of oligomannose glycans in the membrane-bound trimer is consistent with a native-like trimer conformation where glycan processing enzymes have limited accessibility as it has been previously shown (59)(Figure 8). In conclusion, EMPEM analysis revealed several distinct Env sites targeted by serum antibodies of immunized animals whose sera exhibited potent autologous virus neutralization. The polyFab densities mostly targeted sites lacking N-glycans in its respective HIV-1 strain Env (Figure 7). As others have reported for other HIV strains (52, 53), these data suggest that naturally lacking N- glycan sites are likely the main sites of vulnerability for the HIV strains studied here, 1086c and JR-FL.

[0115] This study represents a natural progression on HIV-1 Env design given the success of mRNA vaccines to prevent severe COVID by limiting SARS-2 replication. The advances in HIV Env vaccine design presented here allow us to deliver trimeric Env immunogens by genetic means in the form of mRNA lipid nanoparticles. These designs build on the soluble, cleavage independent Env trimeric platform NFL. As mentioned, this is an advantage in the context of genetic vaccines as interventions on purification of relevant Env forms is not possible. For this, a stabilized form of the NFL trimer design was employed, which was successfully used to make well-ordered nativelike soluble trimers, linking this construct to the natural HIV-1 transmembrane via a small second linker or, alternatively, restoring the membrane proximal external region to complete the gp41 ectodomain. Note, that older non-stabilized NFL designs generate heterogenous oligomers that donot exclusively retain native-like conformations (9, 21). Homogeneity and fidelity to the HIV native Env conformation are key elements for the success of a genetically delivered HIV vaccine. Here, the inventors utilize the most advanced cassette of trimer stabilization design which aims at increasing trimer stability and homogeneity without altering natural antigenic surfaces (10). The inventors generated four NFL.711 cell surface Env constructs from four HIV-1 strains 1086c, BG505, 16055 and JR-FL and created lipid nanoparticles to test the capacity of these new Env mimics to generate a HIV-1 neutralizing antibody response. While the 16055, 1086c and JR-FL NFL.711 membrane-bound trimers delivered as mRNA LNPs generated an autologous neutralizing antibody response, the construct BG505 NFL.711 did not. It was speculated that the weak immunogenicity response from the BG505 NFL construct delivered as mRNA LNPs was due to the limited expression of that Env on the cell surface as was demonstrated that most of that Env is retained inside the cell. The BG505 NFL.711 construct did express adequately from DNA but not from mRNA LNPs. Although the amino acid sequence of these two constructs were identical, the nucleic acid sequence was not, and it is possible that the sequence optimization for mRNA expression reduced translation, translocation to the ER or folding. Indeed, a recent publication demonstrates that modified ribonucleotides can affect fidelity of mRNA translation (60).

[0116] Both matched mRNA and soluble protein immunogens (1086c and JR-FL Env-derived constructs) generated very similar autologous neutralizing antibody responses but, as observed before and by others, each individual animal responded with different magnitude titers in both modalities of the vaccine. The difficulty in penetrating the HIV Env glycan shield might account for the variation between animal antibody responses. The EMPEM analysis, although limited to four animals, suggested that glycan holes are Env regions frequently targeted by B cells in the two HIV strains studied here, 1086c and JR-FL. These results agree with others who have shown similar targets for other HIV strains (52, 53). The EMPEM analysis of the samples derived from soluble protein immunogens but not the mRNA samples did show an additional target in the gp41 interface region (denoted by the yellow density in the right panels of Figure 7) where several glycosylation sites are present suggesting that perhaps the membrane bound Env is glycosylated differently in that region, or that the cell membrane on which the trimers are anchored presents a physical barrier to the elicitation of these gp41 -directed antibodies. The glycan profde analysis seems to support the former explanation as glycan occupancy and composition were especiallydifferent in the trimer base gp41 region. Another interesting observation is that the antibodies targeting the base of Env were present in the mRNA samples, which suggests that at some point, either when the NFL.711 expressing cell is dying or perhaps when trimers are eventually disassembled by proteases the base epitopes are exposed. However, visual inspection of the 2D class averages in the EMPEM analysis revealed that the mRNA LNPs generated less polyfab densities targeting the trimer base than the soluble trimer samples.

[0117] The NFL.711 cell surface expressed HIV-1 trimers described herein represent another tool to generate an HIV-1 vaccine. Genetic expression of HIV Env can facilitate the process of generation of clinical-grade reagents to use in heterologous prime-boosting regimens that will likely be necessary to generate an efficacious HIV vaccine.

[0118] Expression systems and production methods

[0119] The nucleic acids encoding the NFL trimers can be expressed using any suitable expression system. In various embodiments, mammalian expression systems provide native-like glycosylation patterns for proper antigenicity. Alternative expression systems include insect cells, yeast, bacterial systems, or plant-based expression platforms, each offering distinct advantages for specific applications. Production methods encompass both transient and stable expression approaches. Transient expression systems allow for rapid production and testing of different NFL trimer variants, while stable cell lines provide consistent, scalable production for clinical applications. The choice of production method depends on factors including desired scale, timeline, and regulatory requirements.

[0120] HIV envelope glycoprotein (Env)

[0121] The HIV envelope protein (Env) is the target of broadly neutralizing antibodies (bnAbs) in natural infection. Env is a membrane protein composed of a trimer of gp!20 and gp41 subunits that contains a high degree of sequence diversity and a surface that is shielded by N- linked glycans. The bnAbs that target Env often have unusual features such as a long complementarity-determining region (CDR) H3, high levels of somatic hypermutation (SHM), and insertions and deletions (INDELS). Furthermore, most of the bnAbs recognize complex epitopes that are typically non-linear and have both protein and glycan components.

[0122] As used herein, at least three separate zoonotic transmissions resulted in the formation of three distinct HIV-1 groups: M (main), O (outlier), and N (non-M / non-0). About 90% of HIV- 1 infections are classified as group M and these are distributed worldwide. Within the HIV-Mgroup, there is a further division into at least ten subtypes or clades. The immunogens of the present disclosure can be derived from any HIV strain or clade, including but not limited to Clade A (e.g., BG505), Clade B (e.g., JR-FL), and Clade C (e.g., 1086c, 16055).

[0123] Design of cleavage-independent membrane-anchored NFL trimers

[0124] The immunogens disclosed herein are based on a ‘native flexibly linked’ (NFL) design, which renders the Env trimer cleavage-independent, a significant advantage for genetic vaccine approaches. To generate membrane-bound NFL trimers, two primary design strategies were employed. The first strategy involves genetically tethering a soluble, stabilized NFL trimer construct (e.g., truncated at residue 664) to the natural HIV transmembrane (TM) domain via a short, flexible linker, such as a (G4S) linker. This approach bridges the gap created by the absence of the native membrane proximal external region (MPER).

[0125] The second strategy involves restoring the full, natural MPER (e.g., residues 665-683) to the soluble NFL trimer design, thereby creating a full-length Env ectodomain that is then linked to the natural HIV TM and cytoplasmic tail (CT). To improve cell-surface expression and retention, the native long Env CT can be truncated (e.g., at residue G711) to remove endocytosis signals. These designs, referred to herein as NFL.711, result in well-ordered, native-like trimers embedded in the plasma membrane following expression from DNA or mRNA.

[0126] In some embodiments, the constructs of the present invention can have about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to any of the sequences described herein.

[0127] Stabilizing mutations

[0128] To enhance the homogeneity and stability of the expressed trimers, the NFL constructs described herein incorporate a cassette of stabilizing mutations. These mutations were previously developed to generate well-ordered soluble HIV Env NFL trimers and have been shown to transfer effectively to the membrane-bound context. These mutations may include, but are not limited to, those designated as “TD CC+” (Figure 9), which comprises mutations derived from BG505 trimers (Trimer Derived), V3 loop stabilization, Fusion Peptide (FP) stabilization, helix-disrupting mutations (e.g., proline or glycine insertions in gp41), and engineered disulfide bonds to preventCD4-induced conformational changes. The specific combination of stabilizing mutations can be tailored to the specific HTV Env strain being used.

[0129] Nucleic acid constructs

[0130] The various recombinant nucleotide sequences and immunogens of the disclosure may be made using standard recombinant DNA and cloning techniques. The nucleic acid sequences of the present disclosure may be inserted into “vectors.” For the immunogens of the present disclosure to be expressed, the protein coding sequence should be “operably linked” to regulatory or nucleic acid control sequences that direct transcription and translation of the protein. The “nucleic acid control sequence” may be any nucleic acid element, such as, but not limited to promoters, enhancers, internal ribosome entry site (IRES), introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. Any suitable vector may be used depending on the application, for example, plasmids, viral vectors, or other vectors suitable for transient expression in mammalian cells. For example, in the studies described herein, modified gpl40 NFL genes were cloned into a kanamycin-resistant CMVR vector for expression.

[0131] Nucleic acid and mRNA vaccine formulations

[0132] The present disclosure provides for nucleic acids, particularly messenger RNA (mRNA), encoding the immunogenic NFL trimers. For effective delivery and expression in vivo, these nucleic acids are advantageously formulated in nanoparticles, particularly lipid nanoparticles (LNPs). The mRNA sequences may be codon-optimized for enhanced expression in mammalian cells and may include modified nucleosides (e.g., pseudouridine, 5-methylcytidine) to reduce innate immunogenicity and improve stability and translational efficiency.

[0133] The LNP formulations can be optimized for nucleic acid delivery. A typical LNP formulation may comprise an ionizable lipid, a phospholipid, a structural lipid (e.g., cholesterol), and a PEG-modified lipid to prevent aggregation and control particle size. As described in the Examples, the LNPs used had a mean hydrodynamic diameter of approximately 80 nm and an encapsulation efficiency of approximately 95%, properties which are favorable for vaccine delivery.

[0134] Immunogenic compositions and vaccines

[0135] The immunogenic compositions disclosed herein, which may be vaccine compositions, comprise an immunogen as described and a pharmaceutically acceptable carrier or excipient. Thecompositions may be formulated as injectable suspensions, solutions, lyophilized powders, or other suitable forms. To prepare such a composition, a nucleic acid or protein as disclosed herein is mixed with one or more pharmaceutically acceptable carriers, which must be compatible with the other ingredients of the composition. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof.

[0136] Adjuvants may also be included to enhance the immune response. Adjuvants include, but are not limited to, mineral salts (e.g., alum), oil-in-water emulsions (e.g., MF59), saponins (e.g., QS21), and TLR agonists. For example, animals that received soluble protein in the studies described herein received protein adjuvanted with Adjuplex.

[0137] The compositions can be designed to introduce the nucleic acids or expression vectors to a desired site of action. For example, the mRNA immunogens described herein are advantageously formulated in lipid nanoparticles (LNPs) for delivery. The LNP formulation can comprise an ionizable lipid, a PEG-modified lipid, a phospholipid, and a structural lipid such as cholesterol. In the studies disclosed herein, a proprietary LNP formulation was used, as described in US Patent No. 10,221,127, with a mean hydrodynamic diameter of approximately 80 nm and an encapsulation efficiency of approximately 95%.

[0138] Protein production and purification

[0139] Methods which are well known to those skilled in the art may be used to construct expression vectors containing nucleic acid molecules that encode the polypeptides disclosed herein. These methods include in vitro recombinant DNA techniques and synthetic techniques. For example, His-tagged and His-Avi-tagged monomeric and trimeric antigens may be produced by transient transfection of suitable host cells, such as HEK-293F cells, and purified by immobilized metal ion affinity chromatography (IMAC) followed by size-exclusion chromatography (SEC). Nanoparticle immunogens may be produced by transient transfection and purified by lectin affinity chromatography followed by SEC. Monoclonal antibodies may also be produced using transient transfection of host cells and purified using Protein A affinity chromatography. In the studies disclosed herein, soluble trimers were produced in HEK 293F cells, purified by lectin agarose column chromatography, and further purified by size exclusion and negative selection where needed.

[0140] Host cells

[0141] Expression of the immunogens and antibodies may be carried out in any suitable type of host cells. Examples of suitable mammalian host cell lines include but are not limited to CHO, VERO, BHK, HeLa, MDCK, HEK-293T, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e g., COS-1 or COS-7), PER.C6, HsS78Bst, HepG2, SP2 / 0, and TZM- bl cells. In the studies described herein, HEK 293F and HEK 293T cells were used for protein expression and transfection assays.

[0142] Pharmaceutical compositions and formulations

[0143] A pharmacological formulation of the present disclosure may be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the vaccine compositions utilized in the present disclosure may be administered parenterally to the patient in the form of polymer matrices, liposomes, and microspheres.

[0144] Adjuvants are any substance whose admixture with an administered antigen increases or otherwise modifies the immune response to said antigen. Adjuvants may for example be selected from the group consisting of A1K(SC>4)2, AlNa(SO4)2, AINH4 (SO4), silica, alum, Al(0H)3, Cas (PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl- D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'2'- dipalmitoyl-s- n-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene / Tween-80.RTM emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see, e.g., U.S. Pat. No. 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21.

[0145] A pharmacological formulation of the vaccine formulation utilized in the present disclosure may be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and thelike are usable. Known techniques, which deliver the compound orally or intravenously and retain the biological activity, are preferred.

[0146] In one embodiment, a formulation of the present disclosure may be administered initially, and thereafter maintained by further administration. For instance, a formulation of the disclosure may be administered in one type of composition and thereafter further administered in a different or the same type of composition. For example, a formulation of the disclosure may be administered by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition, may be used. In an aspect, the vaccine composition may be administered as a single dose, or the vaccine may incorporate set booster doses.

[0147] The quantity to be administered will vary for the patient being treated and whether the administration is for treatment or prevention. For a live viral vector vaccine, the dosage is typically measured in plaque-forming units (PFU). In an embodiment, the dosage ranges from about 2xl02PFU to about 2x108PFU.

[0148] For any composition to be administered to an animal or human, including the components thereof, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response, such as by titrations of sera and analysis thereof for antibodies or antigens, e.g., by ELISA and / or neutralization assays. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. In an aspect, the time for sequential administrations may be ascertained without undue experimentation. For instance, dosages may be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0149] Thus, the skilled artisan may readily determine the amount of the immunogenic composition and optional additives, vehicles, and / or carrier in compositions and to be administered in methods of the current disclosure.

[0150] Typically, an adjuvant or additive is commonly used as 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, mostpreferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein.

[0151] Examples of compositions which may comprise the immunogenic composition of the disclosure include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0152] Compositions of the disclosure are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the disclosure may be in the "solid" form of pills, tablets, capsules, caplets and the like, including "solid" preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. If nasal or respiratory (mucosal) administration is desired, compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers may preferably dispense a metered dose or, a dose having a particular particle size.

[0153] Compositions of the disclosure may contain pharmaceutically acceptable flavors and / or colors for rendering them more appealing, especially if they are administered orally. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like (e.g., for transdermal administration) and will typically contain a sufficient amount of a thickening agent sothat the viscosity is from about 2,500 to 6,500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity preferably of 2,500 to 5,000 cps, since above that range they become more difficult to administer. However, above that range, the compositions may approach solid or gelatin forms, which are then easily administered as a swallowed pill for oral ingestion.

[0154] Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection or orally. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.

[0155] The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form).

[0156] Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the active compound. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and / or flavors may also be present. The compositions may be isotonic, i.e., it may have the same osmotic pressure as blood and lacrimal fluid.

[0157] The desired isotonicity of the compositions of this disclosure may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

[0158] Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methyl cellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected As long asamount is used that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0159] A pharmaceutically acceptable preservative may be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.

[0160] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert with respect to the delivery vehicle. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems may be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0161] It is generally envisaged that the compositions of the disclosure will be administered by injection, as such compositions are to elicit a protective immune response against a virus. The skilled artisan may, from this disclosure and the knowledge in the art, formulate compositions identified by herein methods for administration by injection and administer such compositions by injection.

[0162] The inventive compositions of this disclosure are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally, the pH may be from about 3 to 8.5. Compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals may be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0163] Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequentadministrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0164] In an aspect, the compositions described herein are formulated as pharmaceutical compositions suitable for administration to a subject. Such compositions comprise an effective amount of, for example, a vaccine vector and a pharmaceutically acceptable carrier, diluent, or excipient. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of immunogenic compositions.

[0165] In an embodiment, the pharmaceutical composition is an injectable formulation. Such formulations are often liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection are also prepared. The preparation is, in an aspect, L-forms, or is emulsified. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.

[0166] In an embodiment, the vaccine is formulated in a buffer, for example, a Tris buffer (e.g., lOmM Tris, pH 7.2). The formulation includes a stabilizer, such as rice-derived recombinant human serum albumin (rHSA, e.g., 2.5mg / mL). In an embodiment, the formulation or a diluent used therewith contains a cryoprotectant or stabilizer such as sucrose (e.g., 10%). In an aspect, the vaccine composition contains no preservatives. Additional components are optionally included, such as antimicrobial preservatives, antioxidants, chelating agents, and buffers to enhance stability, sterility, and isotonicity. For example, isotonic agents such as sugars or sodium chloride are included.

[0167] The compositions described herein are stored under conditions that maintain stability and potency, for instance, at temperatures of -60°C to -80°C or colder. Stability of the immunogenic compositions at different temperatures and dilutions is a consideration for deployment.

[0168] In an aspect, formulations of the immunogenic compositions include alternative stabilizers, adjuvants, or excipients to enhance stability, reduce reactogenicity, or modify the immune response. In an embodiment, formulations are developed for improved thermal stability, potentially reducing cold chain requirements. In some aspects, alternative routes of administration,such as intranasal or subcutaneous, are explored for specific applications or target populations, following appropriate preclinical and clinical evaluation.

[0169] In an aspect, developments in manufacturing processes for the immunogenic compositions described herein include, for example, optimizations for increased scale, yield, consistency, or purity. In an aspect, production utilizes bioreactor systems with volumes ranging from 10L to 10,000L. In an aspect, continuous manufacturing processes are employed. In an aspect, alternative cell lines are used including human cell lines, insect cell systems, or plant-based expression systems. In an aspect, human cell lines include HEK-293 or PER.C6 cells. In an aspect, scale-up involves single-use bioreactor technology. In an aspect, perfusion culture systems are used. In an aspect, microcarrier-based suspension cultures increase yield. In an aspect, advanced formulations incorporate stabilizers including trehalose, mannitol, or specialized protein stabilizers. In an aspect, lyophilized presentations are developed for improved thermostability. In an aspect, nanoparticle delivery systems are employed. In an aspect, formulations are optimized for storage temperatures ranging from -80°C to +25°C. In an aspect, stability periods extend from 6 months to 5 years. In an aspect, additional or refined analytical techniques are employed for in- depth characterization and quality control of the vaccine product.

[0170] Methods of use and administration

[0171] Disclosed herein are methods for eliciting an immune response against HIV in a subject. A "subject" may be any mammal, but is preferably a human, for example, a human that is at risk of infection with HIV-1. The method comprises administering an effective amount of an immunogenic composition as disclosed herein to the subject.

[0172] When provided prophylactically, the immunogenic compositions are ideally administered to a subject in advance of HIV infection or evidence thereof. When provided therapeutically, the immunogenic compositions can serve to ameliorate AIDS symptoms. The compositions can be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, and subcutaneous delivery. In the studies described herein, immunizations in rabbits were administered via the intramuscular route.

[0173] Immunization schedules can be readily determined and may include a single dose or a prime-boost regimen. The methods disclosed herein may include one or more priming immunizations followed by one or more boosting immunizations. For example, in the studies described herein, rabbits were immunized four times at 0, 4, 12 and 24 weeks. An effective amountwill vary, but for mRNA-LNP vaccines, dosages of about 5 pg to 100 pg are contemplated. In the studies disclosed herein, animals received 5 pg of mRNA per dose or 30 pg of protein per dose.

[0174] The immunogenic compositions disclosed herein may be administered orally, subcutaneously, or parenterally including intravenous, intraarterial, intramuscular, and intranasal administration, as well as intrathecal and infusion techniques. Advantageously, the administration is intramuscular. The doses may be single doses or multiple doses over a period of several days. Suitable regimes for initial administration and further doses or for sequential administrations also are variable, and may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan. The quantity to be administered will vary for the patient being treated and whether the administration is for treatment or prevention, and will vary from a few micrograms to a few milligrams for an average 70 kg patient.

[0175] Clinical applications and populationsThe immunogenic compositions find application across diverse clinical scenarios. Prophylactic vaccination represents a primary application, with formulations optimized for healthy populations at risk of HIV exposure. Therapeutic applications may involve different dosing regimens or combination approaches with other HIV interventions. Different populations may benefit from tailored approaches. Pediatric formulations might employ adjusted doses or alternative delivery routes. Immunocompromised populations could require enhanced formulations or modified administration schedules. Geographic considerations might influence strain selection or combination approaches targeting locally prevalent HIV variants.

[0176] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0177] The present invention will be further illustrated in the following Examples, which are given for illustration purposes only and are not intended to limit the invention in any way.ExamplesExample 1: Design of the membrane-bound HIV Env NFL.711 trimer constructs

[0178] The Env sequences to generate NFL.711 constructs were derived from the following sequences stored at the GenBank sequence databank (1086c Genbank Accession Number FJ444395; JR-FL Genbank Accession Number U63632; BG505 Genbank Accession NumberDQ208458; 16055 Genbank Accession Number EFl 17268). The cell surface NFL.711 Env protein design strategy included the replacement of the natural HIV leader sequence for the CD5 leader sequence in the plasmid DNA constructs or for the HLA-DR leader in the mRNA constructs. The four-residue furin cleavage site REKR (residues 508- 511 of HIV Env gp!40) were replaced by a flexible linker (G4S)2 following the NFL design. Two separate design strategies were then utilized to link the gpl40 residue D664 to the natural HIV transmembrane domain (TM). In the first strategy, a second small linker (G4S) was used to span the gap between residue D664 and residue K683 of the gp41 domain. In the second strategy, the inventors restored the natural membrane proximal external region (MPER) from residue D664 to K683. Both design strategies are followed by the natural HIV TM (residues L684 to S703) and a truncated cytoplasmic tail (CT). The CT was truncated at residue G711 before the endocytosis signal to increase retention of membranebound trimeric Env proteins. The CT truncation at residue G711 is what gives the name to the membrane bound construct NFL.711. To all these general design modifications, the inventors then added individual stabilizing modification for each HIV Env as shown in Figure 9 following stabilization cassettes previously used for the soluble stabilized NFLs (9, 10). These sequences are shown in Table 1 herein.Generation and purification of Soluble HIV Env NFL.664 trimers

[0179] Soluble trimers (for ELISAs, immunizations and EMPEM) were derived from the following GenBank databank sequences (1086c Genbank Accession Number FJ444395; JR -FL Genbank Accession Number U63632; BG505 Genbank Accession Number DQ208458; 16055 Genbank Accession Number EFl 17268) to generate stabilized NFL.664 (TD CC+) trimers as described in the past (9, 10). In brief, modified gp!40 NFL genes were ordered from GenScript and cloned into a kanamycin resistant CMVR vector (NIH). A CD5 leader sequence was added at the N-terminus of the env NFL gene to allow secretion of the trimeric proteins into the supernatant. HEK 293F cells were transfected using fectin and after four days of incubation at 37°C in a rotating incubator, supernatants were collected and filtered. Supernatants were loaded overnight onto a lectin agarose column and affinity purified. Following lectin purification, trimers were further purified by size exclusion and negative selection if needed (16). The sequences of these constructs are shown in Table 1 herein.Transfections of envelope glycoproteins from plasmid DNA and mRNA

[0180] A day before the transfection, approximately 8 x 106HEK 293T cells were seeded in a 150 mm tissue culture dish in DMEM, 10% FBS and 1% pen-strep buffer. The next day, plated cells were transfected with DNA plasmids encoding 1086c, BG505, JR-FL and 16055 NFL.711 constructs. DNA was mixed in Opti-mem buffer (Invitrogen) with the transfection reagent Fugene6 at 1 :3 ratio with 5 pg of plasmid DNA per 1 x 106cells and incubated at RT for 20 minutes. The plasmid DNA / Fugene6 mix was then added dropwise to plated cells. Cells were incubated at 37°C and collected 48-72 hours after the transfection for FACS analysis.

[0181] For the mRNA transfections, plated HEK 293T cells were harvested and aliquoted in four separate wells in a 96 well-plate at a density of 3 x 106cells per well. Then, 5 pg of mRNA encapsulated in LNPs were dropped directly on to the cells without any transfecting reagent. The cells were incubated in the 96-well plate for 3 hours at 37°C to facilitate the transfection maximizing the contact between cells and the LNPs. Finally, cells were transferred to a 100 mm culture dish where they were cultured for 48 hrs. After 2 days the cells were harvested and prepared for flow cytometry analysis. mRNA design and production

[0182] The amino acid sequence of 1086c (Genbank Accession Number FJ444395); JR-FL (Genbank Accession Number U63632); BG505 (Genbank Accession Number DQ208458); 16055 (Genbank Accession Number EFl 17268) were obtained as above.Table 1: Amino Acid Sequences of HIV Env NFL Constructs

[0183] The sequences underwent codon optimization and GC enrichment using an algorithm to improve expression and reduce potential immunogenicity of the in vitro transcribed mRNA. The codon optimized sequences were gene synthetized by GenScript with an optimized secretion signal, cloned into an in vitro transcription template containing an optimized T7 promoter, 3’UTR, 5’UTR and a 100-adenine tail. The nucleoside modified mRNA sequences were prepared using the MegaScript transcription kit (ThermoFisher Scientific), cotranscriptionally capped using the CleanCap™ system (TriLink Biotechnologies) and purified using a modified cellulose base chromatography method, precipitated, eluted in nuclease free water, and quantified using the NanoDrop One system. Length and integrity were determined using denaturing agarose gel. Endotoxin content was measured using the GenScript Toxisensor chromogenic assay, and values were below detection levels (0.1 EU / mL). mRNA was frozen at -20oC until formulation.Production and characterization of mRNA-LNP vaccines

[0184] The LNP formulation used in this study is proprietary to Acuitas Therapeutics; 165 the proprietary lipid and LNP composition are described in US Patent No. 10,221,127. The RNA- loaded and empty particles were characterized and subsequently stored at -80°C at an RNA concentration of 1 pg / pl and total lipid concentration of 30 pg / pl. The hydrodynamic size, poly dispersity index (PDI) and zeta potential of mRNA-LNPs were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The mRNA encapsulation efficiency of LNP were determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen). The mean hydrodynamic diameter of mRNA-LNPs was approximately 80 nm with a poly dispersity index of0.02-0.06 and an encapsulation efficiency of approximately 95%. Endotoxin levels were determined using the Limulus Amebocyte Lysate (LAL) chromogenic assay found to be <0.5 endotoxin unit (EU) / mL.Animal immunization and sampling

[0185] New Zealand white female rabbits were purchased and housed at ProSci, Inc. Six groups of five rabbits were immunized bilaterally over the hips with a total volume of 200 pL per site. Briefly, the mRNA LNPs were kept frozen at -80°C for long term storage. The day of inoculation the LNPs were allowed to thaw and diluted in PBS for inoculation. Animals received 5 pg of mRNA per dose. Animals that received soluble protein received 30 pg of protein adjuvanted with Adjuplex (20% v / v) (Sigma- Aldrich).Antigenicity of the cell surface NFL. 711 proteins by FACS analysis

[0186] HEK 293T cells expressing NFL.711 trimeric proteins were harvested 72 hours after DNA plasmid or mRNA LNP transfection by pipetting. Cells were filtered through a 0.7 pm filter. Cells were washed twice with FACS staining buffer and aliquoted to a 96 round-well plate (approximately 1 to 3 million cells per well). In a separate 96 well plate, selected antibodies were added at starting concentration of 25 pg / mL and serially diluted five-fold down the plate six times (Vol. 100 pL per well). The antibody dilutions were transferred from the antibody dilution plate to the plate containing the harvested cells and mixed. The antibody / cells mix was incubated at 4°C for one hour on a rotating platform or lightly shaking so that the cells would not settle. Then, the cells were washed three times with FACS buffer. Secondary anti-human PE conjugated antibody was added at a 1 :200 dilution (vol. 100 pL per well). Cells were incubated with a secondary antibody for 1 hour at 4°C. Cells were washed three times with FACS buffer to remove excess or unbound antibodies. The samples were then run in a Novacyte Flow cytometer. Mean fluorescence Intensity (MFI) values were annotated corresponding to each antibody dilution binding to cells expressing the membrane-bound NFL trimeric proteins and plotted as a data point on that antibody dilution series to make the binding curves shown in figures 2 and 3. These experiments were done several times and two of the experiments are reported in the main figures with standard error of the means.Pseudovirus production

[0187] HEK293 T cells were seeded 24 hours before the transfection at a density of 3 x 106per T-75 flask. Plasmids encoding Env corresponding to the HIV / SIV strains to be tested were cotransfected in HEK 293T cells with an Env-deficient backbone plasmid (pSG3DENV) using Fugene6 at a 1:3 DNA:Fugene6 ratio, 15 pg of the backbone and 5 p of Env per T-75 flask. Cell media containing pseudoviruses were harvested 72 hours after the transfection and stored at -80°C. Pseudoviruses are titrated prior to use in the neutralization assay to determine the appropriate dilution of the virus and the need for dextran. Dextran increases infectivity and is used with viruses that produce lower luciferase signals.Neutralization Assay

[0188] Neutralization activity of the serum and purified IgG samples were carried out in TZM- bl target cells as previously described (27). Briefly, l* 104TZM-bl cells were plated per well in a tissue culture 96 well plate the day before (vol. 100 pL of complete DMEM). In a separate U- bottom plate, the samples (serum or IgG) were prepared and diluted accordingly, then distributed to assay plates where they were mixed with the viruses tested. The sera were initially diluted 1 : 10 and, for purified IgG samples the starting concentration of purified IgG was 2000 pg / mL which approximately compares to a 1 :5 dilution of the serum in potency. These quantities (1 : 10 and 2000 pg / mL) are the final concentrations accounting for the 5-fold dilution with the virus when 40 pL of virus is added to the 10 pL of serum or IgG sample per well. These assay plates were then incubated for Ihr at 37°C to allow the antibodies in the serum or IgG samples to interact with the pseudoviral particles. The media of the tissue culture plates containing the TZM-bL cells was aspirated carefully as to not disturbed the adhered cells (alternatively, the 2* 104cells per well can be added on the day of the assay). Following the aspiration of the media, the serum antibody -virus mix was transferred to the TZM-bL cell culture plates and incubated for Ihr at 37°C to facilitate viral infection. After that incubation, fresh media was added to the cells (on top of the antibody virus mix) and the cells were incubated for 48 hours. Media was removed from the cell plates after 48 hours. The cells were lyzed for 20 min in a rocking platform. Then cell plates containing the lysates were taking to a NE02M (BioteK) instrument where the luciferase substrate was added. Luciferase signals are provided in RLUs then converted to serum neutralization activity and reported as ID50s, inhibitory serum dilution that resulted in 50% of inhibition of viral entry.Similarly, the purified serum IgG neutralization assays were reported as IC50s, inhibitory IgG concentration that mediates 50% of inhibition of viral entry.Purification of IgG from Serum

[0189] 231 In a 12 mL conical tube, 1 mL of Protein A slurry was added and washed with PBS twice by centrifugation at 3000 x g and carefully aspirating the supernatant to remove any traces of ethanol or other preservatives. After washing the agarose beads, 5 ml of PBS and 1 mL of serum sample were added and mixed; then, rocked at RT for 1 hour so that the IgG would bind the Protein A. On a rack, an equal number of disposable columns as serum samples were prepared for purification. The diluted sample-agarose mix was added to a column by pipetting the entire contents of the tube into the column. The sample was allowed to flow through the column while retaining the agarose beads containing the bound IgG. The column was washed by adding 5 mL of 0.5 M NaCl PBS to the column. The bound IgG was eluted by adding 5 mL of IgG elution buffer (Thermos Scientific Ref. 21009) and the acidic pH neutralized by adding 1 M Tris, pH 9 (100 pL / ml of eluate). The eluate containing the IgG was buffered exchanged to PBS in an Amicon Ultra centrifugal filter device (30K) and adjusted to a concentration of 10 mg / mL to normalize the IgG sample concentrations in preparation for the neutralization assay.Immunoprecipitation and gel electrophoresis of membrane bound NFL trimers

[0190] Approximately 8 x 106HEK 293T cells were seeded in a 150 mm tissue culture dish in DMEM, 10% FBS and 1% pen-strep buffer. The next day, plated cells were transfected with DNA plasmids encoding 1086c, BG505, IR-FL and 16055 NFL.711 constructs. Cells were collected 72 hours after the transfection and pelleted at 500 x g for 5 min. Then, the cells expressing the cell surface trimeric proteins were washed once with PBS and allocated to 4 different tubes in 1 mL of 0.5% Triton X-100 PBS. Cells were incubated in 0.5% Triton X-100 PBS to dislodge the cell membrane and release the membrane proteins. The cell debris was pelleted by centrifugation at 12000 x g and carefully collected the supernatant containing the solubilized trimeric proteins. VRC01, PGT145, 10E8 and Fl 05 were added to a final concentration of 25 pg / mL separately to each cell aliquot and let it incubate for 1 hour at 4°C. 40 pL of Protein A beads were added to the sample and rocked at 4°C for 30 mins. Then the protein A beads were washed three times with 0.5 M NaCl PBS to remove unbound proteins. Beads were pelleted by centrifugation and excesssupernatant removed. At this point, the gel loading buffer was added, subjected the samples at 100°C for 3 mins to denature the proteins and then the denatured samples were loaded into a Bolt™ 4-12% Bis-Tris Plus gel (Invitrogen ref. NW04120BOX) and run at 150 V for 35 min.ELISA binding assays

[0191] The day before the assay, 96 well plates were coated with 2 pg / mL (Vol 100 pl per well) of the specific soluble antigen, soluble NFL trimeric proteins matching the immunogen received by the animal whose sera samples are being tested. The next day, after washing the coated plates with PBS, blocking buffer was added (Vol. 300 pL per well, 2% milk, 5% FBS PBS) and incubated the plates at RT for 2 hours. The plates were then washed three times with PBS 0.05% Tween by adding 300 pL of wash buffer to each well. Then, the serum samples were added and serially diluted (starting from 1 :50 six times 5-fold dilutions). After an hour incubation at 37 °C, the plates were washed three times with wash buffer. Secondary antibody (goat anti-rabbit Fc fragment) was added (100 pL per well) at a 1 :2000 dilution. The plates were incubated at 37°C for 30 min and then washed 3 times. Then the substrate (3, 3’, 5, 5’ -tetramethylbenzidine (TMB) was added (100 pL per well). Each plate was allowed to develop for 2 min and then the reaction was stopped by adding (100 pL per well) of 0.16 M H2SO4 to each well. Colorimetric reactions were read in a BioteK NE03M spectrophotometer.Intracellular staining of transfected HEK 293T cells

[0192] HEK 293T cells that were transfected with 5 pg of NFL.711 encoding mRNA LNPs following the method described in this section. After 48 hours in culture at 37°C, the protein transport inhibitor containing Brefeldin A (BD, GolgiPlug) was added at a concentration of 1 pL / mL of cell culture. The cells were incubated in the presence of the inhibitor for 3 hours at 37°C. Cells were harvested by gently scraping them off and placed in FACS staining buffer containing Fc block receptor (FcgII / III receptors, BD Fc Block Cat. No. 553142) for 15 min at 4°C. Cells were washed and permeabilized with the fixation / permeabilization solution (BD, Cytofix / Cytoperm kit, Cat. No. 554714). Permeabilized cells were washed twice with the kit provided wash buffer before proceeding with antibody staining.EMPEM analysis

[0193] IgG was isolated as described above (Purification of IgG from Serum). Papain (Sigma Aldrich) was used to digest IgG to polyclonal antigen-binding fragments (polyFab). Trimer- polyFab complexes were prepared and incubated overnight by mixing 15 pg of 1086c or JR-FL NFL.664 trimer with 1 mg of Fab mixture (containing Fc and residual papain). On the next day, the complexes were purified using a Superdex 200 Increase 10 / 300 GL gel filtration column (Cytiva). Purified complexes were concentrated and diluted to a final concentration of 0.03 mg / mL, which were adsorbed on glow-discharged carbon coated copper mesh grids and stained with 2% (w / v) uranyl formate. Electron microscopy images were collected on an FEI Tecnai TF20 equipped with an TVIPS TemCam F416 CMOS camera (120 keV, 1.68 A / pixel) using the Leginon automated collection software, and processed using Relion 3.0 following standard 2D and 3D classification procedures. UCSF Chimera was used to generate the composite maps, and the representative maps with identified epitopes have been deposited to the Electron Microscopy Data Bank.Gfycan 295 profiling of NFLs

[0194] DeGlyPHER (28) is used to ascertain site-specific glycan occupancy and processivity on the examined glycoproteins. Proteinase K treatment and deglycosylation of Env samples HIV Env glycoprotein (when membrane bound, denatured in 6 M urea) was exchanged to water using Microcon Ultracel PL-10 centrifugal filter. Glycoprotein was reduced with 5 mM tris(2- carboxyethyl)phosphine hydrochloride (TCEP-HC1) and alkylated with 10 mM 2- Chloroacetamide in 100 mM ammonium acetate for 20 min at room temperature (RT, 24°C). Initial protein-level deglycosylation was performed using 250 U Endo H for 5 pg trimer, for 1 h at 37°C. Glycoprotein was digested with 1 :25 Proteinase K (PK) for 30 min at 37°C. PK was denatured by incubating at 90°C for 15 min, then cooled to RT. Peptides were deglycosylated again with 250 U Endo H for 1 h at 37°C, then frozen at -80°C and lyophilized. 100 U PNGase F was lyophilized, resuspended in 20 pl 100 mM ammonium bicarbonate prepared in H218O, and added to the lyophilized peptides. Reactions were then incubated for 1 h at 37°C, subsequently analyzed by LC-MS / MS.LC-MSMS

[0195] Samples were analyzed on a Fusion Lumos mass spectrometer. Samples were injected directly onto a 25 cm, 100 pm ID column packed with BEH 1.7 pm Cl 8 resin. Samples were separated at a flow rate of 300 nL / min on an EASY-nLC 1200 UHPLC. Buffers A and B were 0.1% formic acid in 5% and 80% acetonitrile, respectively. The following gradient was used: 0- 25% B over 120 min, an increase to 40% B over 40 min, an increase to 100% B over another 10 min and 10 min at 100% B for a total run time of 180 min. Column was re-equilibrated with solution A prior to the injection of sample. Peptides were eluted from the tip of the column and nanosprayed directly into the mass spectrometer by application of 2.8 kV at the back of the column. The mass spectrometer was operated in a data dependent mode. Full MSI scans were collected in the Orbitrap at 60,000 resolution. The most abundant ions per scan over a set cycle time of 3 s, were selected for HCD at 30 NCE and MS / MS scans were collected in the Orbitrap at 7,500 resolution. Dynamic exclusion was enabled with exclusion duration of 10 s and ions with charge state +2 to +7 were included.Data Processing

[0196] Protein and peptide identification were done with Integrated Proteomics Pipeline (IP2). Tandem mass spectra were extracted from raw files using RawConverter (29) and searched with ProLuCID (30) against a database comprising UniProt reviewed (Swiss-Prot) proteome for Homo sapiens (UP000005640), UniProt amino acid sequences for Endo H (P04067), PNGase F (Q9XBM8), and Proteinase K (P06873), amino acid sequences for the examined proteins, and a list of general protein contaminants. The search space included no cleavage-specificity. Carbamidomethylation (+57.02146 C) was considered a static modification. Deamidation in presence ofH218O (+2.988261 N), GlcNAc (+203.079373 N), oxidation (+15.994915 M) and N- terminal pyroglutamate formation (-17.026549 Q) were considered differential modifications. Data was searched with 50 ppm precursor ion tolerance and 50 ppm fragment ion tolerance. Identified proteins were filtered using DTASelect2 (31) and utilizing a target-decoy database search strategy to limit the false discovery rate to 1%, at the spectrum level (32). A minimum of 1 peptide per protein and no tryptic end per peptide were required and precursor delta mass cut-off was fixed at 15 ppm. Statistical models for peptide mass modification (modstat) were applied. Census2 (33) label-free analysis was performed based on the precursor peak area, with a 15 ppm precursor mass tolerance and 0.1 min retention time tolerance. “Match between runs” was used tofind missing peptides between runs. Data analysis using GlycoMSQuant (28) was implemented to automate the analysis. GlycoMSQuant summed precursor peak areas across replicates, discarded peptides without NGS, discarded misidentified peptides when N-glycan remnant-mass modifications were localized to non-NGS asparagines and corrected / fixed N-glycan mislocalization where appropriate.

[0197] The invention is further described by the following numbered paragraphs:1. A non-naturally occurring protein comprising a native flexibly linked (NFL) trimer of an HIV-1 envelope glycoprotein (Env), wherein the NFL is linked to a natural HIV transmembrane domain (TM) and a cytoplasmic tail (CT), and wherein the protein is stabilized by a cassette of stabilizing mutations.2. The protein of paragraph 1, wherein the Env is derived from an HIV strain selected from 1086c (GenBank accession number FJ444395), JR-FL (GenBank accession number U63632); BG505 (GenBank accession number DQ208458), or 16055 (GenBank accession number EFl 17268).3. The protein of paragraph 1, wherein the Env is linked to the HIV TM domain via a flexible linker.4. The protein of paragraph 3, wherein the flexible linker has the sequence (648)2.5. The protein of paragraph 1, wherein the Env is linked to the HIV TM domain via a membrane proximal external region (MPER).6. The protein of paragraph 5, wherein the MPER extends from residue D664 to K683.7. The protein of paragraph 1, wherein the CT is truncated at residue G711.8. The protein of paragraph 1, wherein the protein comprises the amino acid sequence of 1086c NFL.711 (-MPER) as set forth in SEQ ID NO: 1.9. The protein of paragraph 1, wherein the protein comprises the amino acid sequence of 1086c NFL.711 (+MPER) as set forth in SEQ ID NO: 2.10. The protein of paragraph 1, wherein the protein comprises the amino acid sequence of 16055 NFL.711 as set forth in SEQ ID NO: 3.11. The protein of paragraph 1, wherein the protein comprises the amino acid sequence of BG505 NFL.711 as set forth in SEQ ID NO: 4.12. The protein of paragraph 1, wherein the protein comprises the amino acid sequence of JR-FL NFL.711 as set forth in SEQ ID NO: 5.The protein of paragraph 1, wherein the protein has about 95% sequence identity to the amino acid sequence of 1086c NFL.71 1 (-MPER) as set forth in SEQ ID NO: 1 , 1086c NFL.711 (+MPER) as set forth in SEQ ID NO: 2, NFL.711 as set forth in SEQ ID NO: 3, BG505 NFL.711 as set forth in SEQ ID NO: 4, or JR-FL NFL.711 as set forth in SEQ ID NO: 5. The protein of paragraph 1, wherein the cassette of stabilizing mutations comprises one or more mutations categorized as Trimer Derived (TD) mutations selected from from E47D, K49E, V65K, E106T, I165L, E172V, T308R, E429R, R432Q, K500R, L543N, N553S, K588R, or E662A. The protein of paragraph 1, wherein the cassette of stabilizing mutations comprises one or more mutations categorized as V3 loop stabilizing mutations selected from N302Y or T320M. The protein of paragraph 1, wherein the cassette of stabilizing mutations comprises one or more mutations categorized as Fusion Peptide (FP) stabilizing mutations selected from F519R or L520R. The protein of paragraph 1, wherein the cassette of stabilizing mutations comprises one or more mutations categorized as helix-disrupting mutations in gp41 selected from I559P, L568G, T569G, or N636G. The protein of paragraph 1, wherein the cassette of stabilizing mutations comprises one or more engineered disulfide bonds. The protein of paragraph 18, wherein the engineered disulfide bond is at positions 1201C and A433C. The protein of paragraph 1, wherein the cassette of stabilizing mutations comprises the addition of an N-glycan. The protein of paragraph 24, wherein the N-glycan is added at K160N or K334T. A nucleic acid encoding the protein of any one of paragraphs 1-21. The nucleic acid of paragraph 22, wherein the nucleic acid is formulated in a nanoparticle. The nucleic acid of paragraph 23 wherein the nanoparticle is a lipid nanoparticle (LNP).A method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the protein of any one of paragraphs 1-21. The method of paragraph 25, wherein the animal is a mammal. The method of paragraph 26, wherein the mammal is a human. A method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the nucleic acid of any one of paragraphs 23-24. The method of paragraph 28, wherein the animal is a mammal. The method of paragraph 29, wherein the mammal is a human. A non-naturally occurring mRNA encoding the protein of any one of paragraphs 1- 21. The mRNA of paragraph 31 wherein the mRNA is formulated in a nanoparticle. The mRNA of paragraph 32 wherein the nanoparticle a lipid nanoparticle (LNP). A method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA of any one of paragraphs 31-33. The method of paragraph 34, wherein the animal is a mammal. The method of paragraph 35, wherein the mammal is a human. The protein of any one of paragraphs 1-21 for eliciting an immune response by a method comprising systemically administering to an animal in need thereof an effective amount of the protein. The protein for eliciting an immune response according to paragraph 37, wherein the animal is a mammal. The protein for eliciting an immune response according to paragraph 38, wherein the mammal is a human. The nucleic acid of any one of paragraphs 22-24 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the nucleic acid. The nucleic acid for eliciting an immune response according to paragraph 40, wherein the animal is a mammal.42. The nucleic acid for eliciting an immune response according to paragraph 41, wherein the mammal is a human.43. The mRNA of any one of paragraphs 31-33 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA.44. The mRNA for eliciting an immune response according to paragraph 43, wherein the animal is a mammal.45. The mRNA for eliciting an immune response according to the preceding paragraph, wherein the mammal is a human.46. Use of protein of any one of paragraphs 1-21 for eliciting an immune response by a method comprising systemically administering to an animal in need thereof an effective amount of the protein.47. Use of protein according to paragraph 46, wherein the animal is a mammal.48. Use of protein according to paragraph 47, wherein the mammal is a human.49. Use of the mRNA of any one of paragraphs 31-33 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA.50. Use of the mRNA according to paragraph 49, wherein the animal is a mammal.51. Use of the mRNA according to paragraph 50, wherein the mammal is a human.* * *

[0198] Reference is made to the documents under “References”, infra. “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989), US Pat. No. 9,796,774, US Pat. No. 11,203,617, US Pat. No. 10,058,604, US Pat. No. 10,221,217, US Pat. No. 10,421,789, US Pat. No. 10,870683, W02010107939, WO2011109511, WO2011109488, W02012030904, W02016205704, WO2016065252, WO2017165674, W02019014405, W02020023827, W02020113199, W0202011774, WO2021222706, and WO2024044684, the entireties of which are incorporated herein by reference, including to illustrate that from the foregoing teachings, it is within the ambit of the skilled artisan to practice, and / or make and use the subject matter of the claims, the inventions of the claims, including embodiments and / or constructs of the presentinvention; the incorporation by reference of this paragraph and the citation of documents of this paragraph that are publicly available are provided as description in this disclosure.

[0199] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.References1. Wyatt R, Sodroski J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science. 1998;280(5371): 1884-8.2. Chen B. Molecular Mechanism of HIV-1 Entry. Trends Microbiol. 2019;27(10):878-91.3. Binley JM, Sanders RW, Clas B, Schuelke N, Master A, Guo Y, et al. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp !20 and gp41 subunits is an antigenic mimic of the trimeric virion associated structure. J Virol. 2000;74(2):627-43.4. Sanders RW, Derking R, Cupo A, Julien JP, Yasmeen A, de Vai N, et al. 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Claims

WHAT IS CLAIMED IS:

1. A non-naturally occurring protein comprising a native flexibly linked (NFL) trimer of an HIV-1 envelope glycoprotein (Env), wherein the NFL is linked to a natural HIV transmembrane domain (TM) and a cytoplasmic tail (CT), and wherein the protein is stabilized by a cassette of stabilizing mutations.

2. The protein of claim 1, wherein the Env is derived from an HIV strain selected from 1086c (GenBank accession number FJ444395), JR-FL (GenBank accession number U63632); BG505 (GenBank accession number DQ208458), or 16055 (GenBank accession number EFl 17268).

3. The protein of claim 1, wherein the Env is linked to the HIV TM domain via a flexible linker.

4. The protein of claim 3, wherein the flexible linker has the sequence (G4S)2.

5. The protein of claim 1, wherein the Env is linked to the HIV TM domain via a membrane proximal external region (MPER).

6. The protein of claim 5, wherein the MPER extends from residue D664 to K683.

7. The protein of claim 1, wherein the CT is truncated at residue G711.

8. The protein of claim 1, wherein the protein comprises the amino acid sequence of1086c NFL.711 (-MPER) as set forth in SEQ ID NO: 1.

9. The protein of claim 1, wherein the protein comprises the amino acid sequence of 1086c NFL.711 (+MPER) as set forth in SEQ ID NO: 2.

10. The protein of claim 1, wherein the protein comprises the amino acid sequence of 16055 NFL.711 as set forth in SEQ ID NO: 3.

11. The protein of claim 1, wherein the protein comprises the amino acid sequence of BG505 NFL.711 as set forth in SEQ ID NO: 4.

12. The protein of claim 1, wherein the protein comprises the amino acid sequence of JR-FL NFL.711 as set forth in SEQ ID NO: 5.

13. The protein of claim 1, wherein the protein has about 95% sequence identity to the amino acid sequence of 1086c NFL.711 (-MPER) as set forth in SEQ ID NO: 1, 1086c NFL.711(+MPER) as set forth in SEQ ID NO: 2, 16055 NFL.711 as set forth in SEQ ID NO: 3, BG505 NFL.71 1 as set forth in SEQ ID NO: 4, or JR -FL NFL.711 as set forth in SEQ ID NO: 5.

14. The protein of claim 1, wherein the cassette of stabilizing mutations comprises a Trimer Derived (TD) mutation selected from E47D, K49E, V65K, E106T, I165L, El 72V, T308R, E429R, R432Q, K500R, L543N, N553S, K588R, or E662A.

15. The protein of claim 1, wherein the cassette of stabilizing mutations comprises one or more mutations categorized as V3 loop stabilizing mutations selected from N302Y or T320M.

16. The protein of claim 1, wherein the cassette of stabilizing mutations comprises one or more mutations categorized as Fusion Peptide (FP) stabilizing mutations selected from F519R or L520R.

17. The protein of claim 1, wherein the cassette of stabilizing mutations comprises one or more mutations categorized as helix-disrupting mutations in gp41 selected from I559P, L568G, T569G, or N636G.

18. The protein of claim 1, wherein the cassette of stabilizing mutations comprises a disulfide bond.

19. The protein of claim 18, wherein the disulfide bond spans the amino acids at positions 201 and 433.

20. The protein of claim 1, wherein the cassette of stabilizing mutations comprises addition of an N-glycan.

21. The protein of claim 20, comprising the N-glycan at position 160 or 334.

22. A nucleic acid encoding the protein of any one of claims 1-21.

23. The nucleic acid of claim 22, wherein the nucleic acid is formulated in a nanoparticle.

24. The nucleic acid of claim 23 wherein the nanoparticle is a lipid nanoparticle (LNP).

25. A method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the protein of any one of claims 1-21.

26. The method of claim 25, wherein the animal is a mammal.

27. The method of claim 26, wherein the mammal is a human.

28. A method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the nucleic acid of any one of claims 23-24.

29. The method of claim 28, wherein the animal is a mammal.

30. The method of claim 29, wherein the mammal is a human.

31. A non-naturally occurring mRNA encoding the protein of any one of claims 1 -21 .

32. The mRNA of claim 31 wherein the mRNA is formulated in a nanoparticle.

33. The mRNA of claim 32 wherein the nanoparticle a lipid nanoparticle (LNP).

34. A method for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA of any one of claims 31-33.

35. The method of claim 34, wherein the animal is a mammal.

36. The method of claim 35, wherein the mammal is a human.

37. The protein of any one of claims 1-21 for eliciting an immune response by a method comprising systemically administering to an animal in need thereof an effective amount of the protein.

38. The protein for eliciting an immune response according to claim 37, wherein the animal is a mammal.

39. The protein for eliciting an immune response according to claim 38, wherein the mammal is a human.

40. The nucleic acid of any one of claims 22-24 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the nucleic acid.

41. The nucleic acid for eliciting an immune response according to claim 40, wherein the animal is a mammal.

42. The nucleic acid for eliciting an immune response according to claim 41, wherein the mammal is a human.

43. The mRNA of any one of claims 31-33 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA.

44. The mRNA for eliciting an immune response according to claim 43, wherein the animal is a mammal.

45. The mRNA for eliciting an immune response according to the preceding claim, wherein the mammal is a human.

46. Use of protein of any one of claims 1-21 for eliciting an immune response by a method comprising systemically administering to an animal in need thereof an effective amount of the protein.

47. Use of protein according to claim 46, wherein the animal is a mammal.

48. Use of protein according to claim 47, wherein the mammal is a human.

49. Use of the mRNA of any one of claims 31-33 for eliciting an immune response comprising systemically administering to an animal in need thereof an effective amount of the mRNA.

50. Use of the mRNA according to claim 49, wherein the animal is a mammal.

51. Use of the mRNA according to claim 50, wherein the mammal is a human.

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