HIV-1 immunogen compositions and methods of using the same

Abstract

Provided herein are immunogenic compositions and vaccines related to State-1-stabilized HIV-1 Env trimers. Methods of use are also provided related to invoking an immune response, for example, to State-1-stabilized HIV-1 Env trimers.

Description

HIV-1 IMMUNOGEN COMPOSITIONS AND METHODS OF USING THE SAMEFIELD

[0001] Immunogenic compositions and vaccines related to HIV-1, and methods of using the same are disclosed in the present application.RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 650,233 filed on May 21, 2024, which is incorporated by reference in its entirety herein.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated by reference in its entirety. Said XML copy, created on May 21, 2025, is named 91016-422356_SeqListing.xml and is 10,900 bytes in size.BACKGROUND

[0004] The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) trimer ((gpl20 / gp41)3) on the surface of the viral membrane. CD4 binding drives the metastable Env from a “closed,” pretriggered (State- 1) conformation through a default intermediate (State-2) conformation to an “open,” CD4-bound prehairpin intermediate (State 3). Binding of the State-3 Env to the CCR5 or CXCR4 coreceptor results in the formation of a highly stable gp41 six-helix bundle, which promotes the fusion of the viral and cell membranes.

[0005] As the only virus- specific molecule on the virion surface, Env is a major target for host antibodies. Env’s heavy glycosylation, strain-dependent sequence variability, conformational flexibility and structural lability are thought to contribute to the ability of HIV-1 to evade the host antibody response. During natural infection, high titers of antibodies are elicited that recognize the conformationally flexible gpl60 Env precursor and disassembled Envs (shed gpl20, gp41 six-helix bundles). These antibodies are poorly neutralizing (pNAbs) because they fail to recognize the mature functional Env trimer, which mainly resides in a State- 1 conformation. After several years of infection, broadly neutralizing antibodies (bNAbs), most of which recognize the pretriggered (State- 1) Env conformation, are elicited in some HIV- 1 -infected individuals. Passively administered monoclonal bNAbs are protective in animal models of HIV-1 infection (Hessell, A.J. et al.,Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat Med 15, 951-954 (2009); Mascola, J.R. et al., Protection of macaques against pathogenic simian / human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J Virol 73, 4009-4018 (1999); Mascola, J.R. et al., Protection of macaques against vaginal transmission of a pathogenic HIV-l / SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 6, 207-210 (2000); Parren, P.W. et al., Antibody protects macaques against vaginal challenge with a pathogenic R5 simian / human immunodeficiency vims at serum levels giving complete neutralization in vitro. J Virol 75, 8340-8347 (2001)). Unfortunately, bNAbs have not been efficiently and consistently elicited in animals immunized with current Env vaccine candidates.

[0006] During natural HIV-1 infection, conformationally dynamic, asymmetric Env trimers may divert the evolution of potentially neutralizing antibodies that need to engage State- 1 Env trimers at precise angles-of-approach. For example, Env changes that stabilize sgpl40 SOSIP.664 trimers do not necessarily stabilize the pretriggered conformation of membrane Envs. Conversely, several changes that have been found to stabilize the pretriggered (State- 1) conformation of membrane HIV- 1 Envs involve Env regions that are heterogeneous or disordered in sgpl40 SOSIP.664 trimer structures. Thus, efforts to stabilize the State- 1 conformation of membrane Envs have proceeded empirically. In that process, several natural Env amino acid polymorphisms that individually stabilize the pretriggered (State-1) conformation of the functional HIV-1 membrane Env trimer have been observed. The pretriggered (State- 1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gpl20 / gp41)3) is targeted by virus entry inhibitors and broadly neutralizing antibodies (bNAbs). Natural polymorphisms in HIV-1 Env have been identified that can modulate the stability of the pretriggered (State- 1) conformation and thereby regulate the triggerability of the functional Env trimer.

[0007] Thus, a modified HIV-1 Env trimer that is stabilized in State- 1 is desirable.SUMMARY

[0008] There are provided immunogenic compositions, comprising: (a) a modified human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) trimer, comprising: (i) three modified HIV-1 Env protomers, wherein each of the three modified HIV-1 Env protomers comprise the amino acid sequence of SEQ ID NO: 1; (ii) three modified HIV-1 Env protomers, wherein each of the three modified HIV-1 Env protomers comprise the amino acid sequence of SEQ ID NO: 2; or(iii) three modified HIV-1 Env protomers, wherein each of the three modified HIV-1 Env protomers comprise the amino acid sequence of SEQ ID NO: 3; or (b) a nucleic acid sequence encoding a modified HIV-1 Env protomer, comprising: (i) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1; (ii) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; or (iii) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3.

[0009] There are provided immunogenic compositions: (a) a modified human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) protomer sequence, comprising: (i) a modified HIV-1 Env protomers comprising the amino acid sequence of: (1) SEQ ID NO: 1; (2) SEQ ID NO: 2; or (3) SEQ ID NO: 3; or (ii) a nucleic acid sequence encoding a modified HIV-1 Env protomer, comprising: (1) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1; (2) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; or (3) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3; and (b) a pharmaceutically acceptable carrier.

[0010] There are provided vaccines, comprising: (a) a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) protomer sequence, comprising: (i) a modified HIV-1 Env protomer comprising the amino acid sequence of: (1) SEQ ID NO: 1; (2) SEQ ID NO: 2; or (3) SEQ ID NO: 3; or (ii) a nucleic acid sequence encoding a modified HIV-1 Env protomer, comprising: (1) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1; (2) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; or (3) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3; and (b) a pharmaceutically acceptable carrier.

[0011] There are provided immunogenic compositions, comprising: (a) a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) means for stabilizing Env trimer State- 1; and (b) a pharmaceutically acceptable carrier.

[0012] There are provided immunogenic compositions, comprising: (a) a nucleic acid sequence encoding a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) means for stabilizing Env trimer state- 1; and (b) a pharmaceutically acceptable carrier.

[0013] There are provided vaccines, comprising: (a) a modified human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer state- 1; and (b) a pharmaceutically acceptable carrier.

[0014] There are provided vaccines, comprising: (a) a nucleic acid sequence encoding a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer state- 1; and (b) a pharmaceutically acceptable carrier.

[0015] There are provided methods of treating human immunodeficiency virus-1 (HIV-1), comprising administering: (a) immunogenic compositions disclosed herein, or (b) vaccines disclosed herein, in a subject with or at risk of having HIV-1.

[0016] There are provided uses of (a) immunogenic compositions disclosed herein, or (b) vaccines disclosed herein, for treating human immunodeficiency virus- 1 (HIV-1).

[0017] There are provided (a) immunogenic composition disclosed herein, or (b) vaccines disclosed herein for use in therapy.

[0018] There are provided (a) immunogenic composition disclosed herein, or (b) vaccines disclosed herein for use in treating human immunodeficiency virus- 1 (HIV-1 ).

[0019] There are provided methods of invoking an immune response, comprising administering: (a) immunogenic compositions disclosed herein, or (b) vaccines disclosed herein, in a subject with or at risk of having HIV- 1.

[0020] There are provided methods of generating antibodies in a subject, comprising administering: (a) immunogenic compositions disclosed herein, or (b) vaccines disclosed herein, in a subject with or at risk of having HIV-1.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows embodiments of phenotypes of HIV-1AD8 Env variants. Env processing, subunit association, gpl20-trimer association, and ability to mediate cell-cell fusion and virus infectivity are shown for the wt and variant HIV-1 AD8 Envs, normalized to those values observed for the wt HIV-1 AD8 Env. Values are colored according to the key, based on their fold increase or decrease compared with those of the wt HIV-1AD8 Env. The results shown are the means and standard deviations derived from at least two independent experiments.

[0022] FIGS. 2A-2B show embodiments of effects of CD4-mimetic compounds and cold exposure on the infectivity of H1V-1AD8 Env variants and primary HIV-1 strains.

[0023] (FIG. 2A) The 50% inhibitory concentrations (IC50 values) of the CD4-mimetic compounds (CD4mcs), BNM-III-170 and CJF-III-288, are reported in pM. Cold sensitivity is reported as the half-life (in days) of the infectivity of pseudotyped viruses incubated on ice. In the CD4mc+cold inhibition assay, 100 pM BNM-III-170 or 10 pM CJF-III-288 was incubated with the pseudotyped viruses for 1 h at 37 °C. The CD4mc-virus mixtures were then incubated on ice for various lengths of time and used to infect Cf2Th-CD4 / CCR5 cells. The half-lives (in days) of infectivity of the viruses are shown. The infectivity of viruses with the wt AD8 Env was reduced tonear-background levels by incubation with the CD4mcs for 1 h at 37°C. The results shown are the means and standard deviations derived from at least two independent experiments. Values are colored according to the key. (FIG. 2B) For viruses pseudotyped with the indicated Envs, the halflife (in days) of the infectivity of viruses after incubation on ice is plotted against the CJF-III-288 IC50 (|iM) on a log-log scale. The parental primary HIV-1 strains used to generate the State-1- stabilized Envs in this study are designated with stars. The wt H1V-1AD8 Env (gray star-) and the State- 1 -stabilized HIV-1AD8 Env mutants (gray dots) are shown. In this plot, the upper-right quadrant is populated by the Env variants with greater State-1 stability (i.e., lower Env triggerability). The State- 1 -associated phenotypes of most of the stabilized HIV-1 AD8 Env mutants (gray dots) exceed those of a panel of representative diverse primary HIV-1 strains (black dots and black / gray stars).

[0024] FIGS. 3A-3D show embodiments of processing, virion incorporation, and gpl20 shedding of Tri FPPR Bam Env variants.

[0025] (FIG. 3A) HEK293T cells were transfected with the pNL4-3.AD8 Bam plasmid containing an infectious HIV-1 provirus with the indicated changes in the AD8 Env. “Bam” indicates the presence of S752F / I756F changes in the Env cytoplasmic tail that prevent gp41 clipping by the HIV- 1 protease in the virus particles. Seventy-two hours after transfection, the cell supernatants were collected, filtered through a 0.45-pm membrane and centrifuged at 14,000 x g for 1 h at 4°C.Precipitated particles and clarified cell lysates were Western blotted with a goat anti-gpl20 antibody, the 4E10 anti-gp41 antibody, and mouse anti-p24CA serum. (FIG. 3B) Tri Bam and Tri FPPR Bam virus particles, purified as described in (FIG. 3A) but at 100,000 x g, were resuspended in IX PBS and incubated with BNM-III-170 at the indicated concentrations, for 1 h at room temperature (RT) or 1 or 2 days on ice. Virus particles were then pelleted and the supernatants containing shed gpl20 were incubated with Galanthus nivalis lectin (GNL) beads for 2 h at room temperature. Beads were washed and the captured proteins were Western blotted with a goat anti-gpl20 antibody. The percentage of shed gpl20 Env relative to the input gpl20 on the virus particles is plotted in the graphs. (FIG. 3C) Shedding of gpl20 from virus particles with Tri FPPR Bam Env and Tri FPPR Bam Env mutants after a 1-day incubation on ice with the indicated concentrations of BNM-III-170 was analyzed as described in (FIG. 3B). (FIG. 3D) Levels of processing and virion incorporation of Tri Bam Env and Tri FPPR Bam Env mutants are reported. The maximum percentage of gpl20 shed after incubation of the virions with 200 pM BNM-III-170 at 0°C for one day is reported in the column on the right. Values are colored according to their fold change relative to those of the TriFPPR Bam virus. Desirable phenotypes (i.e., an increase in cleaved Env or a reduction in gpl20 shedding) are colored gray with a star. The results are representative of those obtained in at least two independent experiments, with the means and standard deviations reported in (FIG. 3D).

[0026] FIGS. 4A-4F show embodiments of the relationship of Env State- 1 stability to other HIV-1 properties. (FIG. 4A) The relationship is shown between the infectious half-life (tl / 2) of viruses pseudotyped with the indicated Envs and gpl20 shedding from the virus particles following exposure to 100 pM BNM-III-170 + cold (0°C). (FIGS. 4B-4E) The State- 1 stability index of each Env variant was calculated by multiplying the CJF-III-288 IC50 values (in pM) by the half-life (in days) of virus infectivity at 0°C (on ice). Correlations between the State- 1 stability index and other HIV-1 properties are shown. (FIG. 4F) Correlation between the anti-CD4 antibody SIM.2 IC50 and the relative infectivity of viruses pseudotyped with the indicated Envs is shown. Spearman rankorder coefficient (rS) and two-tailed probability (P) are reported. The names of the HIV-1AD8 Env mutants are abbreviated as follows: TF - Tri FPPR; AE - K59A / N136E; AA - K59A / T138A; AR - K59A / K574R; ER - N136E / K574R; WR - A316W / K574R; AER - K59A / N136E / K574R; AWR - K59A / A316W / K574R.

[0027] FIGS. 5A-5D show embodiments of sensitivity of viruses with State- 1 -stabilized Envs to neutralization by sCD4-Ig and antibodies. (FIG. 5A) Recombinant luciferase-expressing viruses pseudotyped with the indicated Env variants were evaluated for sensitivity to sCD4-Ig and antibody neutralization. The 50% inhibitory concentration (IC50 values) of the Env ligands are reported in pg / mL. (FIG. 5B) Assays measuring neutralization of the viruses pseudotyped with wt AD8 Env or Tri FPPR valiant Envs by a CD4BS bNAb (VRC01), MPER bNAbs (2F5 and 10E8.v4), and V3 pNAbs (19b, 39F and 447-52D) are shown. (FIG. 5C) In an assay to measure the ability of antibodies to neutralize cell-free viruses, recombinant viruses pseudotyped with the indicated Envs were incubated for 1 h at 37° with the following concentrations of antibodies: VRC01 (10 pg / mL), 2F5 and 10E8.v4 (20 pg / mL), 19b, 39F and 447-52D (50 pg / mL). The viruses were then pelleted and the antibody-containing supernatants removed. The virus pellet was resuspended in antibody- free medium and incubated with TZM-bl cells in the presence of 20 pg / ml DEAE-dextran. Fortyeight hours later, luciferase activity was measured. (FIG. 5D) In an assay to measure the ability of antibodies to neutralize virus after virus-cell interaction, recombinant viruses pseudotyped with the indicated Envs were added to TZM-bl cells. The virus-cell mixtures were centrifuged at 1800 x g for 30 min. The medium was removed and replaced with fresh medium containing the antibody. The antibody concentrations were the same as in (FIG. 5C) above. Forty-eight hours later, luciferaseactivity in the TZM-bl cells was measured. For (FIGS. 5A-5D), the means and standard deviations derived from at least two independent experiments are shown.

[0028] FIG. 6 shows embodiments of effects of State- 1- stabilizing changes on the phenotypes of viruses with Envs from clade C and D HIV-1 strains.

[0029] Env processing, subunit association, cell-cell fusing ability and virus infectivity are shown for the wt and variant H1V-1DU422.1 (clade C) and HIV-1191859 (clade D) Envs, normalized to those values observed for the respective wt Env. The sensitivity of viruses pseudotyped with the Env variants to inhibition by the CJF-III-288 CD4mc, exposure to cold (0°C), or combined exposure to CJF-TIT-288 + cold is shown, as detailed in the legend to FIGS. 2A-2B. Values are colored according to the key, based on the fold increase or decrease compared with those of the respective wt HIV-1 Env. Note that the wt Du422.1 and 191859 Envs have a glutamine residue at 543, which was retained in the Tri FPPR variants (except for Tri FPPR(N), in which residue 543 is an asparagine).

[0030] FIG. 7 shows embodiments of effects of Env State- 1 stability on HIV-1 infectivity and susceptibility to antibody neutralization.

[0031] A wt HIV-1 Env and a highly State- 1 -stabilized Env are compared in unliganded states or following engagement of the virus with a CD4+ target cell. In the absence of ligands, the Envs of primary HIV-1 largely occupy State 1, but depending on their triggerability, also sample more open conformations, some of which are recognized by pNAbs. State- 1- stabilizing changes limit the spontaneous sampling of conformations recognized by pNAbs and decrease Env triggerability by CD4. Upon the initial engagement with a single CD4 molecule on the target cell, Env exposes V3, CD4i and MPER epitopes. Wild-type HIV-1 Envs either engage additional CD4 molecules or undergo inactivation. Once multiple CD4 molecules are bound, the V3 and CD4i epitopes are sterically occluded by the target membrane, whereas the MPER epitopes remain available for the binding of bNAbs. The transitions of the State-l-stabilized Env after initially binding CD4 are less efficient, slowing establishment of the sterically protected full CD4-bound conformation and decreasing functional inactivation by events such as gpI20 shedding. The prolonged presence of a more stable functional Env intermediate with less-than-complete occupancy by CD4 increases the opportunity for neutralization by V3-directed pNAbs. The efficacy of MPER bNAbs, which also recognize this Env intermediate, is likewise enhanced. The binding of CD4i pNAbs is likely to be sterically blocked even in the case of Env bound to a single CD4 molecule.

[0032] FIG. 8 shows embodiments of phenotypes of HIV-1AD8 Env variants not selected for further assays. The phenotypes of these Env variants were determined using infectious virusesproduced by the pNL4-3.AD8 Bam proviral plasmid. These Env valiants contain the Bam changes (S752F / I756F) in the cytoplasmic tail that decrease cleavage by the HIV-1 protease [SI -S3]. The phenotypes of these Env valiants were determined using a luciferase-expressing HIV-1 vector pseudotyped with the indicated Envs. These Env variants were expressed using the pSVIIIenv plasmid and do not contain the Bam changes in the cytoplasmic tail.

[0033] FIGS. 9A-9D show embodiments of effects of cold and CD4mcs on the infectivity of viruses with HIV-1AD8 Env variants. (FIGS. 9A-9D) The infectivity of recombinant luciferaseexpressing HIV-1 pseudotyped with the indicated HIV-1AD8 Env valiants, HIV-1JR-FL Env, HIV- 1PV0.4 Env or the A-MLV control Env was measured on Cf2Th-CD4 / CCR5 target cells (FIGS. 9A- 9B) or TZM-bl target cells in the presence of 20 pg / ml DEAE-dextran (FIGS. 9C-9D). (FIGS. 9A and 9C) The viruses were incubated at 0°C (on ice) for the indicated times before measuring their infectivity. (FIGS. 9B and 9D) The viruses were treated with 10 pM CIF-III-288 (FIG. 9B) or 100 pM BNM-III-170 (FIG. 9D) for 1 h at 37°C before incubating the virus-CD4mc mixture at 0°C for the indicated times. In (FIGS. 9A-9D), the relative infectivity represents the measured luciferase activity in the target cells, relative to that seen for the same virus not exposed to cold or a CD4mc. In (FIG. 9C) and (FIG. 9D), viruses with the Comb G Env [S4,S5] and Envs from the primary Tier-2 HIV-1JR-FL and Tier-3 HIV-1PV0.4 are included for comparison. In (FIGS. 9A-9D), means and standard deviations from triplicate measurements in an experiment are shown. The experiments were repeated with comparable results.

[0034] FIGS. 10A-10C shows embodiments of CD4mc activation of infection of CD4-negative, CCR5-expressing cells. Recombinant luciferase-expressing HIV-1 pseudotyped with the indicated variants of Envs from HIV-1AD8 (FIG. 10A), Du422.1 (FIG. 10B), or 191859 (FIG. 10C) were incubated at 37 °C with CD4-negative, CCR5-expressing Cf2Th-CCR5 cells in the presence of the indicated concentrations of CIF-III-288. After 48 h, luciferase activity (RLU) in the Cf2Th-CCR5 cells was measured. The means and standard deviations from triplicate measurements in an experiment are shown. The experiment was repeated with comparable results.

[0035] FIG. 11 shows embodiments of relationships between Env State-1 stability and other HIV- 1 phenotypes. Correlations between the HIV-1 phenotypes (left column) and indicators of Env State- 1 stability were evaluated [S6]. The Spearman rank-order correlation coefficient (rS) and two-tailed P value are shown for each potential relationship.

[0036] FIG. 12 shows embodiments of sensitivity of viruses pseudotyped with HIV-1 Env variants to SIM.2a, maraviroc, and pNAbs. Recombinant luciferase viruses pseudotyped with the indicatedEnv variants were incubated with Cf2Th-CD4 / CCR5 target cells in the presence of increasing concentrations of the SIM.2 anti-CD4 antibody [S7] . Luciferase activity in the Cf2Th-CD4 / CCR5 target cells was measured 48 h later. The virus inhibition curves were analyzed using a four- parameter dose-response model in Graph Pad® Prism 9 to obtain the reported IC50 values. ND - not determined.

[0037] Recombinant luciferase-expressing viruses pseudotyped with the indicated Env variants were incubated with various concentrations of maraviroc or pNAbs for 1 h at 37°C. The viruses were then incubated with Cf2Th-CD4 / CCR5 target cells and luciferase activity was measured 48 h later. The virus inhibition curves were analyzed using a four-parameter dose-response model in GraphPad® Prism 9 to obtain the reported IC50 values. ND - not determined.

[0038] FIGS. 13A-13C show embodiments of antigenicity of Tri Bam Env valiants on virus particles. (FIGS. 13A and 13C) HEK293T cells were transfected with the pNL4-3.AD8 Bam proviral plasmid expressing the indicated HIV-1AD8 Env variants. Seventy-two hours later, the cell supernatants were collected, filtered through a 0.45-pm membrane and centrifuged at 100,000 x g for 1 h at 4°C. Purified virus particles were resuspended in lx PBS and incubated with a panel of broadly neutralizing antibodies (bNAbs) and poorly neutralizing antibodies (pNAbs) for 1 h at room temperature. The virus-antibody mixture was diluted 20-fold with lx PBS and centrifuged to remove unbound antibodies. The virus-antibody pellet was lysed and precipitated with protein A-agarose beads for 1 h at 4°C. The beads were washed three times and Western blotted with a goat anti-gpl20 antibody and the 4E10 anti-gp41 antibody. (FIG. 13B) Antigenicity of Tri FPPR Bam Env and its mutants was evaluated as described in (FIG. 13A) and (FIG. 13C) above. However, after the protein A-agarose incubation and washing step, the Env-antibody complexes were denatured and deglycosylated with PNGase F for 1.5 h at 37°C. The samples were then Western blotted with a goat anti-gpl20 antibody. The results are representative of those obtained in at least two independent experiments. The means and standard deviations of the results are reported in the bar graphs in the panels. There were no significant differences in antibody binding to the Env variants, as evaluated by a two-tailed Student's t test.

[0039] FIG. 14 shows embodiments of neutralization of wt and mutant Du422.1 and 191859 viruses by sCD4-Ig and antibodies. Recombinant luciferase-expressing viruses pseudotyped with the indicated Env variants were incubated for 1 h at 37 °C with sCD4-Ig or the antibodies shown. The virus-antibody mixtures were then added to Cf2Th-CD4 / CCR5 cells. After 48 h of culture, luciferase activity in the Cf2Th-CD4 / CCR5 cells was measured. The infectivity measured in the presence ofthe antibody or sCD4-Ig was normalized to that seen in the absence of antibody. The virus neutralization curves were analyzed using a four-parameter dose-response model in GraphPad® Prism 9 to obtain the reported IC50 values.

[0040] FIGS. 15A-15E show embodiments of effects of State-1- stabilizing changes in the HIV- 1JR-FL Env. Effects of the Tri and FPPR changes on the HIV-1JR-FL E168K Env on virus particles were evaluated. The JR-FL Env contains a natural methionine residue at 535, so the FPPR changes consist only of L543Q and A532V. (FIG. 15A) HEK 293T cells were transfected with the pNL4- 3.JR-FL E168K plasmid containing the indicated changes in the JR-FL E168K Env. Seventy-two hours later, virus particles were purified and analyzed by Western blotting with a goat anti-gp!20 antibody, the 4E10 anti-gp41 antibody and mouse anti-p24CA serum. (FIG. 15B) Shedding of gpl20 from JR-FL E168K Env variants after a 1-day incubation on ice in the presence of the indicated concentrations of BNM-III-170 was analyzed as described in Figure 3B. (FIG. 15C) Results from (FIG. 15A) and (FIG. 15B) are quantified. Max shedding value indicates the percentage of shed gpl20 relative to input gpl20 at 200 pM BNM-III-170 after a 1-day incubation on ice. (FIGS. 15D-15E) Antigenicity of JR-FL E168K Env variants on virus particles was analyzed as described in FIGS. 13A and 13C, respectively. The Envs precipitated from the virus particles in (FIG. 15E) were digested with PNGase F before being analyzed by Western blotting. The results are representative of those obtained in at least two independent experiments. The means and standard deviations of the results are reported in (FIG. 15C) and in the bar graphs of (FIG. 15D) and (FIG. 15E). The significance of the difference in antibody binding between the unmodified JR-FL E168K Env and its mutants was evaluated by a Student's t test; *, P < 0.05; **, P < 0.01.

[0041] FIG. 16 shows embodiments of epitopes of V3 and CD4-induced (CD4i) pNAbs on the CD4-bound Env trimer. A composite structural model based on a CD4-bound soluble Env trimer (5THR) and a CD4-bound gpl20 with an intact V3 region (2B4C) is shown from two perspectives. The relationship of the Env binding sites for CD4 Domain 1 (DI) and pNAbs against V3 and CD4- induced (CD4i) gpl20 epitopes is illustrated. The single-chain Fv (scFv) fragment of the 17b pNAb recognizes a CD4i gpl20 epitope. The 17b scFv and V3 structures are shown only on a single protomer for clarity. Both CD4i and V3 gpl20 elements contribute to CCR5 / CXCR4 binding. The schematic diagrams of the CD4-bound Envs in Figure 7 are based on this composite structure and are consistent with recent structural models. Note that, in the CD4-bound conformation, CD4i pNAbs bind roughly parallel to the Env trimer axis. On the other hand, V3 pNAbs potentially utilize more varied angles of attack and thus may better access the State- 1 -stabilized Env intermediate.

[0042] FIG. 17 shows a schematic representation of embodiments of the wild-type (wt) HIV- 1AD8 Env, with the gpl20-gp41 cleavage site depicted as a black triangle. S, signal peptide; VI to V5, gpl20 major hypervariable regions; Cl to C5, gpl20 conserved regions; FP, fusion peptide; HR, heptad repeat region; TM, transmembrane region; CT, cytoplasmic tail. The amino acid numbers beneath the diagram mark some of the key boundaries. The amino acid residue changes in the Env mutants compared to the wt AD8 Env are shown. The Env changes that contribute to the stabilization of the pretriggered Env conformation are highlighted in gray. The degree of stabilization of the pretriggered Env exhibits the order: Tri FPPR-K59A / K574R > Tri FPPR > AE.2 > AD8.

[0043] FIG. 18 shows antibody recognition and immunoprecipitation (IP) of the HIV-1AD8 Tri FPPR Env variants on the cell surface. (A) A549 cells were induced with doxycycline to express the indicated Tri FPPR (TF) Env variants. The cells were detached from the tissue culture plates with 5 mM EDTA in lx PBS. After pelleting and resuspension, the cells were aliquoted and incubated with the indicated antibodies or sCD4-Ig. After washing, the cells were lysed in NP-40 lysis buffer. The clarified cell lysates were incubated with protein A-Sepharose beads. The precipitated antibody-Env complexes were Western blotted with a goat anti-gpl20 antiserum (upper panels) or the human 4E10 anti-gp41 antibody (lower panels). The experiment was repeated, and a typical result is shown. (B) The gpl60, gpl20, and gp41 / gp26 bands in A were quantified using Fiji ImageJ (NIH). For the F240 anti-gp41 antibody only, the intensities of the gp41 / gp26 bands were normalized to those of the respective gp41 / gp26 bands precipitated by the 2G12 antibody. The intensities of the gpl20 bands precipitated by the other antibodies were normalized to those of the respective gpl20 bands precipitated by the 2G12 antibody. The means and standard deviations of the results of two independent experiments are shown. The bNAbs are colored blue (labeled) and the pNAbs are colored red (labeled).

[0044] FIG. 19 shows the comparison of the antigenicity of H1V-1AD8 Env variants solubilized in different amphipathic copolymers. A549 cells were induced with doxycycline to express the wt HIV-1AD8, AE.2, or Tri FPPR Envs. Cell membranes were purified, and the Envs were extracted with the indicated amphipathic copolymers. In half of the samples, BMS-806 was added and maintained at a concentration of 10 pM throughout Env purification. The Envs were purified by Ni- NTA affinity chromatography. A small panel of antibodies was used to assess the antigenicity of the Envs eluted from the Ni-NTA beads. The 19b pNAb (red, labeled) recognizes a gpl20 V3 epitope. The PGT145, PG9, and VRC03 bNAbs (blue, labeled) exhibit some preference for the pretriggered(State- 1) Env conformation. The Env variants precipitated by the antibodies were captured on protein A-Sepharose beads and subjected to Western blotting with a goat anti-gpl20 antiserum. The experiments were repeated with similar results. Quantification of the gpl20 band intensity was performed in Fiji ImageJ (NIH). In most cases, the intensity of the gpl20 band was normalized to that of VRC03-bound gpl20; in cases where the VRC03-bound gpl20 band intensity was less than that of 19b-bound gpl20, normalization was done to the 19b-bound gpl20 band intensity (e.g., wt and AE.2 Envs in SMA and AASTY 11-50, or Tri FPPR Envs in SMA). The means and standard deviations derived from at least two independent experiments are shown. The levels of gpl20 precipitated by the PGT145, PG9, and VRC03 bNAbs were compared with the level of gp!20 precipitated by the 19b pNAb, using a Student t test. Two-tailed P values are indicated (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0045] FIG. 20 shows the antigenicity of the A 18- solubilized wt HIV-1AD8, AE.2 and Tri FPPR Envs after Ni-NTA purification from cell membranes. (A) A549 cells were induced with doxycycline to express the wt HIV-1 AD8, AE.2 and Tri FPPR Envs. Forty-eight hours later, the cells were detached using 5 mM EDTA in lx PBS. Cell membranes were prepared, incubated with BMS-806 or buffer control, and extracted with 0.5% A18. Envs were purified by Ni-NTA affinity chromatography, and the eluates from the Ni-NTA beads were incubated with the indicated antibodies or sCD4-Ig and protein A-Sepharose beads for 1 h at room temperature. The precipitated proteins were Western blotted with a goat anti-gpl20 antiserum (upper panels) and the human anti- gp4I antibody (lower panels). (B) Quantification of the intensity of the gpl20 and gp41 bands in panel A was performed in Fiji ImageJ (NIH). For the F240 anti-gp41 antibody only, the intensities of the gp41 bands were normalized to those of the respective gp41 bands precipitated by the 2G12 antibody. For the other antibodies, the intensities of the gpl20 bands were normalized to those of the respective gpl20 bands precipitated by the 2G12 antibody. The means and standard deviations of the results derived from two independent experiments are shown. The bNAbs are colored blue (labeled), and the pNAbs are colored red (labeled).

[0046] FIG. 21 shows the antigenicity of A 18- solubilized Tri FPPR Env variants after Ni- NTA purification from cell membranes. (A) A549 cells were induced with doxycycline to express the indicated Tri FPPR (TF) Env variants. Forty-eight hours later, the cells were detached using 5 mM EDTA in 1. PBS. The cell membranes were purified using a sucrose cushion and extracted with 0.5% A18 followed by Ni-NTA affinity chromatography. The eluate from the Ni-NTA beads was aliquoted and incubated with the indicated antibodies or sCD4-Ig together with protein A-Sepharosebeads for 1 h at room temperature. An aliquot without added antibody served as the input sample. The input sample and precipitated proteins were Western blotted with a goat anti-gpl20 antiserum (upper panels) and the human 4E10 anti-gp41 antibody (lower panels). (B) Quantification of the intensity of the gpl20 and gp41 / gp26 bands in panel A was performed in Fiji ImageJ (NIH). For the F240 anti-gp41 antibody only, the intensities of the gp41 / gp26 bands were normalized to those of the respective gp41 / gp26 bands precipitated by the 2G12 antibody. For the other antibodies, the intensities of the gpl20 bands were normalized to those of the respective gpl20 bands precipitated by the 2G12 antibody. The means and standard deviations derived from two independent experiments are shown. The bNAbs are colored blue (labeled), and the pNAbs are colored red (labeled).

[0047] FIG. 22 shows the correlation analysis between antibody recognition of cell-surface Envs and A18-solubilized Envs. The correlation is shown between the antigenicities of HIV-1AD8 Tri FPPR (A), TF-K59A / K574R (B), and TF-K59A / K574R A715 (C) Envs on the cell surface and the A 18- solubilized, purified Envs (IP, immunoprecipitation). The antigenicities of the Envs on the cell surface were determined as described in the Fig. 2 legend. The antigenicities of the solubilized Envs in A18 lipid-nanodiscs were determined as described in the Fig. 20 legend. The pNAbs are colored red (triangles), and the bNAbs are colored blue (circles). The Spearman rank correlation coefficient (rS), the Pearson r (rP), and two-tailed P values are shown.

[0048] FIG. 23 shows the stability of A 18- solubilized Tri FPPR Env conformation over time at different temperatures. The stability of the A 18- solubilized Tri FPPR Env trimer was evaluated by measuring its antigenicity after incubation at 4 or 37°C for different times. (A) A549 cells were induced with doxycycline to express the HIV-1AD8 Tri FPPR Env, which has a 6-His tag at the carboxyl terminus. At 48 h after induction, the cells were detached by treatment with 5 mM EDTA. Cell membranes were purified using a sucrose cushion and were lysed in 0.5% A18. BMS-806 (10 pM) was added before A18 solubilization and maintained at this concentration throughout Env purification and analysis of antigenicity. HIV-1 Envs were purified by Ni-NTA affinity chromatography. The eluates were aliquoted and incubated at 4 or 37 °C for either 2 or 7 days. A panel of antibodies [bNAbs (blue, labeled) and pNAbs (red, labeled)] and sCD4-Ig was used to assess the antigenicity of the Tri FPPR Env after the incubation period. Western blotting was performed to analyze the precipitated HIV-1 Envs, which were detected with a goat anti-gpl20 antiserum (upper panels) or the human 4E10 anti-gp41 antibody (lower panels). (B) Quantification of the gpl20 and gp41 band intensity in A was performed in Fiji ImageJ (NIH). For the F240 anti-gp41 antibody only, the gp41 band intensity was normalized to the gp41 band of the VRC03 antibody. For the other antibodies, the gpl20 band intensity was normalized to the gpl20 band precipitated by the 2G12 antibody. The means and standard deviations from two independent experiments are shown. Antibody recognition of the Tri FPPR Env before (Day 0) and after incubation at 4°C or 37°C was compared, using a Student t test. Two-tailed P values indicate significant differences:*? < 0.05; **P < 0.01; ***P < 0.001.

[0049] FIG. 24 shows stability of A 18- solubilized Envs after freezing and thawing in the presence of different cryoprotectants. A549 cells were induced with doxycycline to express the Tri FPPR (A) or TF-K59A / K574R Envs (B). Forty-eight hours later, the cells were detached from the tissue culture plates with 5 mM EDTA in 1. PBS. Cell membranes were purified using a sucrose cushion, and the Envs were extracted with 0.5% A18. The HIV-1 Envs were purified by Ni-NTA chromatography. To assess the effect of cryoprotectants on the integrity of the HIV-1 Envs after freezing and thawing (F-T), the Envs were untreated, frozen and thawed without any cryoprotectants, or frozen and thawed in the presence of the indicated cryoprotectant. A small panel of antibodies was used to assess the antigenicity of the Tri FPPR (A) and TF-K59A / K574R (B) Envs. Western blotting was performed with a goat anti-gpl20 antiserum (upper panels) or the human 4E10 anti-gp41 antibody (lower panels). Quantification of the gpl20 and gp41 band intensity in A was performed in Fiji ImageJ (NIH). For the 19b, PG9, and VRC03 antibodies, the gpl20 band intensity was normalized to that seen for the VRC03 antibody. For the F240 anti-gp41 antibody, the gp41 band intensity was normalized to that seen for the VRC03 antibody. The means and standard deviations from the two independent experiments are shown in (A), and the results of a typical experiment are shown in (B).

[0050] FIG. 25 shows HIV-1 Env conformation on virus-like particles (VLPs) resists the effects of freezing and thawing. (A, upper panels) A549 cells were induced with doxycycline to express defective virus-like particles (VLPs) containing HIV-1 TF-K59A / K574R (117). Two days after induction, the supernatants were harvested and spun at 1,500 . g for 10 min at 4°C and filtered through a 0.45-pm polyvinylidene difluoride (PVDF) membrane. The filtrates were then centrifuged at 100,000 . g for 1 h at 4°C. The pellets were resuspended in 1. PBS and spun at 20,000 . g for 1 h at 4°C. The pellets were resuspended in 1. PBS and divided into three equal parts for treatment by freezing and thawing. One fraction served as a control; a second fraction was subjected to freezing and thawing; 10% sucrose was added to a third fraction before freezing and thawing. (A, lower panels) A549 cells were induced with doxycycline to express full-length HIV-1 Env TF-K59A / K574R or the cytoplasmic tail-truncated HIV-1 Env TF-K59A / K574R A715. Two days after induction, A549 cells were detached using 5 mM EDTA in 1. PBS. The cell membranes were purified using a sucrose cushion, and the Envs were extracted with A18. The Envs were purified using Ni-NTA chromatography and aliquoted for freezing and thawing, as described above. (B) Quantification of the gpl20 and gp41 / gp26 band intensity in A was performed in Fiji ImageJ (NIH). For the F240 anti-gp41 antibody, the gp41 / gp26 band intensity was normalized to the gp41 / gp26 band intensity seen for the VRC03 antibody. For the other antibodies, the gpl20 band intensity was normalized to that seen for the VRC03 antibody. The means and standard deviations from two experiments are shown. The levels of antibody recognition were compared with a Student t test (****, two-tailed P < 0.0001).

[0051] FIG. 26 shows the effect of adjuvants on A18-solubilized HIV-1 Env conformation. (A) To evaluate the effect of different adjuvants on the integrity of A18-solubilized HIV-1 Env, A549 cells were induced with doxycycline to express full-length HIV-1 Env Tri FPPR. Forty-eight hours after induction, the cells were detached with 5 mM EDTA. Cell membranes were purified using a sucrose cushion and were lysed in 0.5% A18. BMS-806 (10 pM) was added before A18 solubilization and maintained at that concentration throughout the subsequent analyses. The Tri-FPPR Env was purified by Ni-NTA affinity chromatography. The purified HIV-1 Env was incubated with different adjuvants at 37°C for 1 h. A small panel of antibodies was used to assess the antigenicity of Env after incubation with adjuvants. Western blotting was performed with a goat anti-gpl20 antiserum (upper panels) or the human 4E10 anti-gp41 antibody (lower panels). (B) Quantification of the gpl20 and gp41 band intensity in A was performed in Fiji ImageJ (NIH). For the F240 anti-gp41 antibody only, the gp41 band intensity was normalized to that seen for the VRC03 antibody. For the other antibodies, the gpl20 band intensity was normalized to that seen for the 2G12 antibody. The results of an experiment in which the highest concentrations of each adjuvant were used are shown. No significant differences in antibody recognition were observed.

[0052] FIG. 27 shows the variables that influence the effect of extraction from membranes on HIV-1 conformation. The charts summarize the effects of extracting the wild-type HIV-1AD8 Env or a State- 1 -stabilized Env (Tri FPPR) from the membranes of expressing cells with SMA, AASTY 11-50 or Al 8 in the absence or presence of BMS-806. The data are derived from the experiment shown in Fig. 19 and represent the average recognition of the solubilized Env by bNAbs relative to that by the 19b pNAb, using the formula [(PGT145 + PG9 + VRC03) 3] / 19b. Theintensity of blue shading is proportionate to the above ratio and represents the degree to which a pretriggered conformation is preserved in the solubilized Env.

[0053] FIG. 28 shows the strategy for establishing cells inducibly producing defective HIV- 1 VLPs with stabilized pretriggered Envs. (A) The def4 and def5 HIV-1 proviruses used in this study are shown. Smaller arrows indicate the positions of stop codons. The 5' stop codon in pol disrupts the open reading frame encoding reverse transcriptase (RT), RNase H, and integrase (IN). Additional stop codons disrupt the expressions of IN and Vif in def4 and def5. Compared with def4, def5 has a stop codon eliminating Vpr expression and two modifications (U3tetOx2 and TARmod) of the long terminal repeat (LTR) that renders transcription dependent on the expression of the reverse tetracycline-regulated transactivator (rtTA) and doxycycline. The env genes encoding the parental AD8 Env and stabilized pretriggered variants were introduced into the def4 and def5 proviruses. The def4 and def5 proviruses retain the wild-type HIV-lNL4-3gag, tat, rev, vpu, and nef genes and encode a functional protease (PR). (B) The strategy for establishing producer cells that inducibly release defective VLPs with stabilized pretriggered Envs is shown. The def5 plasmid is cotransfected with the pCMV-Vpr-RT-IN plasmid and the pCMV-VSV G plasmid into Apobec3g-negative packaging cells constitutively expressing rtTA. In the presence of doxycycline (Dox), these packaging cells produce recombinant viruses that can mediate a single round of infection. Then, the transduced rtTA-expressing producer cells release VLPs with Envs encoded by def5 in response to treatment with doxycycline. The inset shows the structure of a stabilized AE2 Env trimer (PDB: 8FAE). The three Env protomers are colored in shades of blue, red, and white. The gp41 subunits (at the top of the image) are colored in darker shades than the gpl20 subunits (at the bottom of the image). The Tri and FPPR changes that stabilize the pretriggered Env conformation are shown as numbered Corey-Pauling-Coltun (CPK) atoms (dark). Amino acid residues are numbered according to standard convention. The Tri Bam and Tri FPPR Bam Envs used in this study contain the Tri and Tri + FPPR changes, respectively; as a result, the pretriggered conformation of the Tri FPPR Bam Env is more stable than that of the Tri Bam Env.

[0054] FIG. 29 shows a comparison of virus particles produced by transfection of pNL4- 3. env and def4 proviral plasmids. HEK 293T cells were transfected with the pNL4-3.env plasmid or the def4 plasmid expressing the AD8 Bam, Tri Bam, or Tri FPPR Bam Envs. In some samples, a plasmid expressing the Vpr-RT-IN protein was cotransfected at a weight ratio of 50% of that of the proviral plasmids. Seventy-two hours after transfection, the cells were harvested and lysed. The cell supernatants were filtered (0.45 pm), and portions of the filtered supernatants were used for pelletingthe virus particles at 14,000 x g for 1 h at 4°C. (A) The clarified cell lysates and virus pellets were western blotted with a goat anti-gpl20 antibody, the 4E10 anti-gp41 antibody, and the mouse anti- p24 CA antibody. The total amount of gpl20 + gp41 Env in a given volume of cell supernatant and the (gpl20 + gp41):p24 ratio in the cell supernatants relative to those observed for the pNL4-3.env are shown in the bar graphs. The reported means and standard deviations are derived from the virus particles with the three Envs tested. (B) The indicated volumes of the HEK 293T cell supernatants were incubated with TZM-bl cells for 48 h before the cells were lysed, and luciferase activity was measured. The means and standard deviations are from triplicate luciferase readings within a typical experiment. (C) Purified virus particles with the Tri FPPR Bam Env were incubated with a panel of broadly neutralizing antibodies (bNAbs) and poorly neutralizing antibodies (pNAbs) for 1 h at room temperature. The virus-antibody mixture was washed with lx PBS and centrifuged. The virusantibody pellet was lysed and precipitated with protein A-agarose beads for 1 h at 4°C. The beads were washed three times and western blotted with a goat anti-gpl20 antibody and the 4E10 anti- gp41 antibody. The input sample was prepared as described in Example 21. The relative levels of cleaved Env (gpl20 and gp41) on the vims particles, normalized to the levels of gpl20 and gp41, respectively, in the input sample, are shown in the bar graphs on the right. The results of a single experiment are shown; the experiment was repeated with similar results.

[0055] FIG. 30 shows the estimation of Tri FPPR Bam Env content on VLPs from def5 expressing A549 cells. (A) A549 cells transduced with the def5 provirus expressing Tri FPPR Bam Env were treated with 2 pg / mE of doxycycline, and after 48 h, the cell culture supernatant was clarified by centrifugation at 600 x g for 15 min, filtered through a 0.45-pm filter, pelleted by ultracentrifugation at 100,000 x g for 1 h at 4°C, and then solubilized in Laemmli buffer. Samples of the VLP lysates, equivalent to the volumes shown in each image, were run on SDS-PAGE. Purified HIV-1 recombinant gpl40 Env (JR- FL, ARP- 12573) and p24 Gag-GFP (51 kDa) proteins corresponding to the indicated pmol amounts were included as standards. Western blots were prepared and probed with saturating amounts of polyclonal anti-gpl20 (left) and monoclonal anti- p24 (right) antibodies. (B) The signal intensity of each band was quantified and expressed as pixels using UN-SCAN-IT gel analysis software. The pmol amount of each protein standard was plotted against its respective pixel value to generate a standard curve and best-fit linear equation. (C) With each of the western-blotted VLP samples (shown in A), the pixel count of gpl20 and each p24- containing band was determined to calculate molar concentrations in units of pmol / pL. The average value of the 1 and 2 pL samples was used to estimate the relative molar ratio of gpl20 Env to p24Gag on VLPs produced from the def5-expressing A549 cells. (D) In an independent experiment, VLPs from A549 cells transduced with the def5 provirus expressing Tri FPPR Bam Env were prepared as described above in A, except that VLPs were pelleted either through a 20% sucrose cushion (right) or without a sucrose cushion (left). The pellets were solubilized in 300 L of Laemmli buffer. Two-fold serial dilutions of the VLP lysates were prepared and loaded on gels in the amounts shown. The purified recombinant gpl40 and p24-GFP proteins were mixed, serially diluted, and loaded together on SDS-PAGE gels in the pmol amounts indicated for each. Western blots were prepared and probed with polyclonal anti-gpl20 antibody (top panels). After chemiluminescence images were collected, the same blots were reprobed with anti-p24 monoclonal antibody (bottom panels). The signal intensities of the bands were quantified and analyzed to determine VLP gpl20 / p24 molar ratios, using methods described above in B and C.

[0056] FIG. 31 shows cryo-electron tomography of VLPs with Tri FPPR Bam Env. (A) A representative tomogram slice is shown of VLPs with Tri FPPR Bam Envs produced in A549 cells. Envs are indicated by arrows. Scale bar = 100 nm. (B) The frequency distribution of Env numbers on each VLP is shown, n = 70. (C) Sub tomogram- averaged Tri FPPR Bam Env on VLPs is shown in side and top views. (D) The capsid morphologies of VLPs with Tri FPPR Bam Envs produced in A549 cells are shown. Four types of VLPs were observed: (i) empty viral particles (vesicles); (ii) immature-like capsids (immature); (iii) improperly sealed capsids; and (iv) cone-shaped capsids (mature). Representative tomogram slices are shown for each type of VLP, with the colors of the borders corresponding to those in the pie chart. Percentages of each type of VLP are shown in the pie chart. Scale bar = 50 nm. (E) The distribution of the number of Envs on each type of VLP in (D) is shown, n.s. - not significant.

[0057] FIG. 32 shows the characterization of VLPs produced from Dox-induced A549 cells. (A) Cell lysates and virus particles were analyzed from HEK 293T cells transfected with def4 plasmids expressing the indicated Envs and from A549 cells transduced with def5 proviruses with the indicated Envs. The Tri FPPR Bam Flag Env has a FLAG epitope inserted into the gpl20 V4 region between the FNGTWL and TQSNGT sequences. In 12-well plates, 0.9 x 105 HEK 293T cells per well and 1.8 x 105 def5 -transduced A549 cells per well were seeded. As HEK 293T cells grow more rapidly than A549 cells, fewer HEK 293T cells were seeded. Approximately 16-18 h later, the HEK 293T cells were transfected with 1 pg def4 plasmids, and A549 cells were induced with 2 pg / mL doxycycline. Seventy-two hours later, the supernatants were filtered (0.45 pm) and precipitated at 14,000 x g for 1 h at 4°C. The clarified cell lysates and virus pellets were western blotted with a goatanti-gpl20 antibody, the 4E10 anti-gp41 antibody, a mouse anti-p24 CA antibody, and a rabbit anti- hsp70 antibody. The bar graphs on the right show the means and standard deviations of the indicated ratios for the averages of all the Envs in each group (except for Tri FPPR Bam Flag, which because of its decreased processing, was not included in the quantitation). (B) The first three lanes were prepared from def5-transduced A549 cells as described in A. To prepare viruses with the AD8 Bam Env from T cells, C8166-R5 cells were infected with a medium from HEK 293T cells transfected with pNL4-3.AD8 Bam. Infection of the C8166-R5 T cells was carried out at a multiplicity of infection of 1 in the presence of 8 pg / mL polybrene. The C8166-R5 cells were washed the next day, and 4 days later, 75% of the suspension culture was removed and replaced with a fresh medium. The virus-containing supernatant was collected on day 10 after infection, and viruses were purified as described in A and analyzed by western blotting. (C) Viruses from pNL4-3.env-transfected HEK 293T cells or def5 -transduced A549 cells with the indicated Envs were prepared as described above. Equivalent volumes of filtered (0.45 pm) virus-containing supernatants were incubated with C8166- R5 cells. The cells were washed 3 h later. Every 3-5 days following infection, 75% of the suspension culture was removed and replaced with a fresh medium. The virus titer in the medium was measured on TZM-bl cells, as described in the Example 21. The C8166-R5 culture was infected with the AD8 Bam virus from pNL4-3.AD8 Bam-transfected HEK 293T cells succumbed to viral cytopathic effects at day 15 after infection (marked with an X). (D) VLPs with the indicated Envs were prepared from different passages of def5-transduced A549 cells, as described in A. Samples were stored at -20°C before western blotting. (E) Virus particles with AD8 Bam Envs were prepared from def5-transduced A549 cells and infected C8166-R5 T cells, as described in A and C, respectively. Purified virus particles were lysed, followed by treatment with PNGase F or Endo Hf enzymes for 1.5 h at 37°C. Viral lysates were western blotted with a goat anti-gpl20 antibody and the 4E10 anti-gp41 antibody. Envs deglycosylated with PNGase F and Endo Hf are indicated with red (underlined) and green arrows, respectively. (F) Purified virus particles from def5 -transduced A549 cells expressing the indicated Envs were incubated with BS3 crosslinker at the indicated concentrations for 30 min at room temperature. The samples were subsequently quenched, analyzed by reducing SDS-PAGE, and western blotted with a goat anti-gpl20 antibody. Except for C, the results are representative of those obtained in at least two independent experiments. The means and standard deviations of the results from the western blots or from triplicate readings in C arc reported in the graphs. The significance of the difference between samples was evaluated by a Student’s t test; *, P < 0.05; **, P < 0.01.

[0058] FIG. 33 shows env antigenicity and gp 120- shedding efficiency on virus particles produced in A549 cells. (A) A549 cells transduced with def5 proviruses expressing the indicated Envs were incubated with 2 pg / mL doxycycline. Seventy-two hours later, the supernatants were filtered (0.45 pm) and precipitated at 100,000 x g for 1 h at 4°C. Virus pellets were resuspended in lx PBS, and Env antigenicity was evaluated as described in the Fig. 29C legend. Input samples, which were used for normalization of the gpl20 and gp41 signals, were prepared as described in Example 21. (B) A549 cells were transfected using Lipofectamine 3000 with the pNL4-3.AD8 Bam plasmid expressing AD8 Bam Envs with or without the D368R change. The D368R change drastically reduces the ability of Env to bind CD4. At 5-9 days after transfection, the virus was pelleted from culture supernatants, and Env antigenicity was evaluated as described in A. (C) VLPs prepared from def5-transduced A549 cells as in A were incubated with the CD4-mimetic compound BNM-III-170 at the indicated concentrations for 1 h at room temperature. VLPs were then pelleted, and the supernatants containing shed gpl20 were incubated with GNL beads for 2 h at room temperature. Beads were washed and western blotted with a goat anti-gpl20 antibody. The results shown are representative of those obtained in at least two independent experiments. The means and standard deviations of the results are reported in the bar graphs. The significance of the difference in antibody binding or shedding between the AD8 Bam Env and the other Envs was evaluated by a Student’s t test; *, P < 0.05; **, P < 0.01.DETAILED DESCRIPTION

[0059] Provided herein are membrane Env variants stabilized in pretriggered conformations (i.e. , State- 1 -stabilized) beyond that found in natural HIV-1 strains. State-1 stability correlated with stronger trimer subunit association, increased virus sensitivity to bNAb neutralization and decreased capacity to mediate cell-cell fusion and virus entry. Further, in some embodiments, after virus-host cell engagement, the normally inaccessible gpl20 V3 region on an Env intermediate became targetable by otherwise poorly neutralizing antibodies. Non-naturally occurring combinations of naturally occurring State- 1 -stabilizing Env valiants exhibited unpredictable results; in most cases, the combinations of State- 1 -stabilizing changes showed additive effects on State- 1 stabilization; in fewer cases, the combinations resulted in decreases in desirable Env properties (e.g., processing, subunit association). Provided herein are empirically derived, non-naturally occurring membrane Envs that are stabilized in State- 1 conformations beyond those found in natural HIV-1 strains.COMPOSITIONS

[0060] Provided in the present disclosure are immunogenic compositions and vaccines. In embodiments, the immunogen composition comprises a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer. In embodiments, the immunogen composition comprises a nucleic acid sequence encoding a protomer that forms a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer. In embodiments, the vaccine comprises a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer. In embodiments, the vaccine comprises a nucleic acid sequence encoding a protomer that forms a modified human immunodeficiency virus- 1 (HIV- 1) envelope glycoprotein (Env) trimer. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers. In embodiments, the immunogen composition comprises modified HIV-1 Env trimer comprising three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: 1. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: 2. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: 3. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is State-1 stabilized. In embodiments, the modified HIV- 1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is more sensitive to broadly neutralizing antibodies than the parental HIV-1 Env or unmodified HIV-1 Env. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is sensitive to broadly neutralizing antibodies with an IC50 of 2 pg / ml - 1 pg / ml, 2 pg / ml - 1 pg / ml, 1 pg / ml - 0.1 pg / ml, 0.1 pg / ml - 0.01 pg / ml ; 2 pg / ml or lower than 2 pg / ml; 1 pg / ml or lower than 1 pg / ml; or 0.1 pg / ml or lower than 0.1 pg / ml.

[0061] Provided in the present disclosure are immunogenic compositions and vaccines. In embodiments, the immunogenic composition comprises a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer, and a pharmaceutically acceptable carrier. Inembodiments, the immunogenic composition comprises a nucleic acid sequence encoding a promoter that forms a modified human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) trimer, and a pharmaceutically acceptable carrier. (E.g., a nucleic acid sequence that drives expression of RNA encoding Env protomer). In embodiments, the vaccine comprises a modified human immunodeficiency virus-1 (HIV-l) envelope glycoprotein (Env) trimer, and a pharmaceutically acceptable carrier. In embodiments, the vaccine comprises a nucleic acid sequence encoding a promoter that forms a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer, and a pharmaceutically acceptable carrier. (E.g., a nucleic acid sequence that drives expression of RNA encoding Env protomer). In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers. In embodiments, the immunogen composition comprises a modified HIV-1 Env trimer comprising three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: I. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: 2. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: 3. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is State-1 stabilized. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is more sensitive to broadly neutralizing antibodies than the parental HIV-1 Env or unmodified HIV-1 Env. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is sensitive to broadly neutralizing antibodies with an IC50 of 2 pg / ml - 1 pg / ml, 2 pg / ml - 1 pg / ml, 1 pg / ml - 0.1 pg / ml, 0.1 pg / ml - 0.01 pg / ml ; 2 pg / ml or lower than 2 pg / ml; 1 pg / ml or lower than 1 pg / ml; or 0.1 pg / ml or lower than 0.1 pg / ml.

[0062] Provided in the present disclosure are immunogenic compositions and vaccines. In embodiments, the immunogenic composition comprises a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer state- 1 and apharmaceutically acceptable carrier. In embodiments, the immunogenic composition comprises a nucleic acid sequence encoding a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer state- 1 and a pharmaceutically acceptable carrier. In embodiments, the vaccine comprises a modified human immunodeficiency virus- 1 (HIV- 1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer state- 1 and a pharmaceutically acceptable carrier. In embodiments, the vaccine comprises a nucleic acid sequence encoding a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer state- 1 and a pharmaceutically acceptable carrier. In embodiments, the modified HIV-1 Env trimer means for stabilized Env trimer state- 1 comprises three modified HIV-1 Env protomers. In embodiments, the immunogen composition comprises a modified HIV-1 Env trimer comprising three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: I . In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: 2. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein each protomer comprises SEQ ID NO: 3. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is State- 1- stabilized. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is more sensitive to broadly neutralizing antibodies than the parental HIV-1 Env or unmodified HIV-1 Env. In embodiments, the modified HIV-1 Env trimer comprises three modified HIV-1 Env protomers, wherein the modified HIV-1 Env protomers comprise SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the modified HIV-1 Env trimer is sensitive to broadly neutralizing antibodies with an IC50 of 2 pg / ml - 1 pg / ml, 2 pg / ml - 1 pg / ml, 1 pg / ml - 0.1 pg / ml, 0.1 pg / ml - 0.01 pg / ml ; 2 pg / ml or lower than 2 pg / ml; 1 pg / ml or lower than 1 pg / ml; or 0.1 pg / ml or lower than 0.1 pg / ml.

[0063] In embodiments, the immunogenic compositions disclosed herein are stabilized compared with prior wild-type or modified HIV-1 Env trimers. In embodiments, immunogenic compositions of the present disclosure have a half life of 10 days, at least 10 days, or greater than 10 days at 0°C; 11 days, at least 11 days, or greater than 11 days at 0°C; 12 days, at least 12 days, or greater than 12days at 0°C; 13 days, at least 13 days, or greater than 13 days at 0°C; 14 days, at least 14 days, or greater than 14 days at 0°C; 15 days, at least 15 days, or greater than 15 days at 0°C; 16 days, at least 16 days, or greater than 16 days at 0°C; 17 days, at least 17 days, or greater than 17 days at 0°C; 18 days, at least 18 days, or greater than 18 days at 0°C; 19 days, at least 19 days, or greater than 19 days at 0°C; or 20 days days, at least 20 days, or greater than 20 days at 0°C, such as 16 days, at least 16 days, or greater than 16 days at 0°C or 19 days, at least 19 days at 0°C, or greater than 19 days at 0°C.

[0064] In embodiments, the immunogenic compositions disclosed herein are resistant to CD4- mimetic molecules, such as BNM-II1-170 or CJF-III-288, compared with prior wild-type or modified HIV-1 Env trimers. In embodiments, immunogenic compositions of the present disclosure bind CD4-mimetic molecules, such as BNM-III-170 or CJF-III-288, with an IC50 of 100 pM or greater than 100 pM, 125 pM or greater than 125 pM, 150 pM or greater than 150 pM, 175 pM or greater than 175 pM, 200 pM or greater than 200 pM, 225 pM or greater than 225 pM, 250 pM or greater than 250 pM, such as greater than 150 pM (e.g., CJF-III-288 IC50) or greater than 200 pM (e.g., BNM-III-170 IC50). In embodiments, the immunogenic compositions disclosed herein remain stabilized even in the presence of CD4-mimetic molecules. In embodiments, the immunogenic compositions disclosed herein have a half life of 9 days, at least 9 days, or greater than 9 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III- 170; 10 days, at least 10 days, or greater than 10 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170; 11 days, at least 11 days, or greater than 11 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF- III-288 or 100 pM BNM-III-170; 12 days, at least 12 days, or greater than 12 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170; 13 days, at least 13 days, or greater than 13 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-111-288 or 100 pM BNM-111-170; 14 days, at least 14 days, or greater than 14 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF- III-288 or 100 pM BNM-III-170; 15 days, at least 15 days, or greater than 15 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170; 16 days, at least 16 days, or greater than 16 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170; 17 days, at least 17 days, or greater than 17 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF- III-288 or 100 pM BNM-III-170; 18 days, at least 18 days, or greater than 18 days at 0°C even inthe presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170; 19 days, at least 19 days, or greater than 19 days at 0°C; or 20 days days, at least 20 days, or greater than 20 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170, such as 16 days, at least 16 days, or greater than 16 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170or 19 days, at least 19 days at 0°C, or greater than 19 days at 0°C even in the presences of CD4-mimetic molecules, such as 10 pM CJF-III-288 or 100 pM BNM-III-170.

[0065] Provided herein are immunogenic compositions including the disclosed modified HIV-1 Env protomers or trimers, and / or nucleic acids encoding the protomers or trimers are also disclosed herein. In embodiments, the immunogenic compositions can include one or more of pharmaceutically acceptable carriers, adjuvants (such as those described above), a stabilizing detergent (such as polysorbate 80 (TWEEN® 80) (Sorbitan-mono-9-octadecenoate-poly(oxy-l,2- ethanediyl); manufactured by ICI Americas, Wilmington, Del.), TWEEN® 40, TWEEN® 20, TWEEN® 60, ZWITTERGENT® 3-12, TEEPOL® HB7, and SPAN® 85 detergents, for example, in an amount of approximately 0.05 to 0.5%, such as at about 0.2%), a micelle-forming agent (such as PLURONIC® L62LF, L101, and L64 block copolymer, polyethylene glycol 1000, and TETRONIC® 1501, 150R1, 701, 901, 1301, and 130R1 block copolymer, for example, between 0.5 and 10%, or in an amount between 1.25 and 5%), an oil (squalene, squalane, eicosane, tetratetracontane, glycerol, and peanut oil or other vegetable oils, for example, in an amount between 1 and 10%, or between 2.5 and 5%), and a cryoprotectant (sucrose, propanol, methanol, ethanol, DMSO, glycerol, and ethylene glycol). In some embodiments the adjuvants are selected from alhydrogel, poly (I:C), CpG oligodeoxynucleotide (ODN), monophosphoryl lipid A (MPLA) in liposomes, Quil-A, squalene, or a combination thereof. In some embodiments, the immunogenic composition includes a 10% concentration of sucrose or glycerol. In embodiments, the disclosed immunogenic compositions are delivered by styrene-maleic acid lipid nanoparticles (SMALPs), by other amphotropic copolymer-lipid nanodiscs, or by self-assembling protein nanoparticles (SAPNs; e.g., hemagglutinin, human papilloma virus LI major capsid protein, Hepatitis B surface antigen, bacteriophage Q0) and certain bacterial proteins (e.g., ferritin, encapsuling, lumazine synthase) (e.g., e.g., as described in U.S. Pat. Pub. No. 2024 / 0115693, incorporated by reference herein in its entirety on May 21, 2024). In some embodiments, the copolymer-lipid nanodiscs include SMA, AASTY 11-50, and Al 8.

[0066] In embodiments, immunogenic compositions herein comprise nucleic acids encoding a disclosed protomer. In embodiments, a therapeutically effective amount of the nucleic acid(s) is administered to a subject, such as a human or mammalian subject to generate an immune response. In embodiments, a therapeutically effective amount of the nucleic acid(s) is administered to a subject, such as a human or mammalian subject with or at risk of HIV-1, to generate an immune response. In embodiments, immunogenic compositions disclosed herein are administered as nucleic acids encoding a disclosed protomer by direct immunization with plasmid DNA, such as with a mammalian expression plasmid. In embodiments, an expression plasmid encodes a disclosed protomer under the control of a promoter to express the disclosed protomer. Invoking an immune response by nucleic acid constructs is well-known in the ail (e.g., as described in U.S. Pat. Nos: 5,643,578; 5,593,972; and 5,817,637, incorporated by reference herein in their entireties on May 21, 2024). Delivery of nucleic acid constructs for invoking an immune response is well-known in the art (e.g., as described in U.S. Pat. No. 5,880,103, incorporated by reference herein in its entirety on May 21, 2024). Known methods of delivering nucleic acid constructs include liposomes and immune- stimulating constructs (e.g., as described in U.S. Pat. No. 9,855,329, incorporated by reference herein in its entirety on May 21, 2024); attenuated viral hosts or vectors or bacterial vectors (e.g., as described in U.S. Pat. No. 4,722,848 and 9,855,329, incorporated by reference herein in their entireties on May 21, 2024); gold microspheres or direct injection (e.g., intramuscular injection) of plasmids under promoter control (e.g., as described in U.S. Pat. No. 9,855,329, incorporated by reference herein in its entirety on May 21, 2024).Protomers and Sequences of Interest in the Immunogenic Compositions and Vaccines of the Present Disclosure

[0067] In embodiments, the protomer binds with two additional protomers and forms an Env trimer. In embodiments, one, two, or all three protomers comprise SEQ ID NO: 1: MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASD AAAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDeSL KPCVKLTPLCVTLNCTDLRNVTNINNSSEGMRGEIKNCSFNITTSIRDKVKKDYALFYRLDV VPIDNDNTSYRLINCNTSTITQACPKVSFEPIPIHYCTPAGFAILKCKDKKFNGTGPCKNVSTV QCTHGIRPVVSTQLLLNGSLAEEEVVIRSSNFTDNAKNIIVQLKESVEINCTRPNNNTRKSIHI GPGRAFYTTGDIIGDIRQAHCNISRTKWNNTLNQIATKLKEQFGNNKTIVFNQSSGGDPEIVM HSFNCGGEFFYCNSTQLFNSTWNFNGTWNLTQSNGTEGNDTITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLILTRDGGNNHNNDTETFRPGGGDMRDNWRSELYKYKVVKIEPL GVAPTKAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGvASmTLTVQARqLLSGIVQQQN NLLRAIEAQQHLLkLTVWGIRQLQARVLtVERYLRDQQLLGIWGCSGKLICTTAVPWNASW SNKTLDMIWNNMTWMEWEREIDNYTGLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDITNWLWYIKIFIMIVGGLIGLRIVFTVLSIVNRVRQGYSPLSFQTHLPAPRGPDRPEGIEEEGGDRDRDRSVRLVDGSLAL1WDDLRSLCLFSYHRLRDLLL1VTR1VELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 1, particular sequence variations in bold and underlined).

[0068] In embodiments, the immunogenic compositions herein comprise a nucleic acid sequence encoding SEQ ID NO: 1.

[0069] In embodiments, the protomer binds with two additional protomers and forms an Env trimer. In embodiments, one, two, or all three protomers comprise SEQ ID NO: 2:MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDeSL KPCVKLTPLCVTLNCTDLREVTNINNSSEGMRGEIKNCSFNITTSIRDKVKKDYALFYRLDVVPIDNDNTSYRLINCNTSTITQACPKVSFEPIPIHYCTPAGFAILKCKDKKFNGTGPCKNVSTV QCTHGIRPVVSTQLLLNGSLAEEEVVIRSSNFTDNAKNIIVQLKESVEINCTRPNNNTRKSIHI GPGRAFYTTGDIIGDIRQAHCNISRTKWNNTLNQIATKLKEQFGNNKTIVFNQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNFNGTWNLTQSNGTEGNDTITLPCRIKQIINMWQEVGKA MYAPPIRGQIRCSSNITGLILTRDGGNNHNNDTETFRPGGGDMRDNWRSELYKYKVVKIEPL GVAPTKAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGvASmTLTVQARqLLSGIVQQQNNLLRAIEAQQHLLkLTVWGIRQLQARVLtVERYLRDQQLLGIWGCSGKLICTTAVPWNASW SNKTLDMIWNNMTWMEWEREIDNYTGLIYTLIEESQNQQEKNEQELLELDKWASLWNWF DITNWLWYIKIFIMIVGGLIGLRIVFTVLSIVNRVRQGYSPLSFQTHLPAPRGPDRPEGIEEEGGDRDRDRSVRLVDGSLAL1WDDLRSLCLFSYHRLRDLLL1VTR1VELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 2, particular sequence variations in bold and underlined).

[0070] In embodiments, the immunogenic compositions herein comprise a nucleic acid sequence encoding SEQ ID NO: 2.

[0071] In embodiments, the protomer binds with two additional protomers and forms an Env trimer. In embodiments, one, two, or all three protomers comprise SEQ ID NO: 3:MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASD AAAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDeSL KPCVKLTPLCVTLNCTDLREVTNINNSSEGMRGEIKNCSFNITTSIRDKVKKDYALFYRLDV VPIDNDNTSYRLINCNTSTITQACPKVSFEPIPIHYCTPAGFAILKCKDKKFNGTGPCKNVSTV QCTHGIRPVVSTQLLLNGSLAEEEVVIRSSNFTDNAKNIIVQLKESVEINCTRPNNNTRKSIHI GPGRAFYTTGD11GD1RQAHCN1SRTKWNNTLNQ1ATKLKEQFGNNKT1VFNQSSGGDPE1VM HSFNCGGEFFYCNSTQLFNSTWNFNGTWNLTQSNGTEGNDTITLPCRIKQIINMWQEVGKA MYAPPIRGQIRCSSNITGLILTRDGGNNHNNDTETFRPGGGDMRDNWRSELYKYKVVKIEPL GVAPTKAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGvASmTLTVQARqLLSGIVQQQN NLLRAIEAQQHLLkLTVWGIRQLQARVLtVERYLRDQQLLGIWGCSGKLICTTAVPWNASW SNKTLDMIWNNMTWMEWEREIDNYTGLIYTLIEESQNQQEKNEQELLELDKWASLWNWF DITNWLWYIKIFIMIVGGLIGLRIVFTVLSIVNRVRQGYSPLSFQTHLPAPRGPDRPEGIEEEGG DRDRDRSVRLVDGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNL LQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 3, particular sequence variations in bold and underlined).

[0072] In embodiments, the immunogenic compositions herein comprise a nucleic acid sequence encoding SEQ ID NO: 3.

[0073] In embodiments, the protomer binds with two additional protomers and forms an Env trimer. In embodiments, one, two, or all three protomers comprise modified SEQ ID NO: 4: MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASD AKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDESL KPCVKLTPLCVTLNCTDLRNVTNINNSSEGMRGEIKNCSFNITTSIRDKVKKDYALFYRLDV VPIDNDNTSYRLINCNTSTITQACPKVSFEPIPIHYCTPAGFAILKCKDKKFNGTGPCKNVSTV QCTHGIRPVVSTQLLLNGSLAEEEVVIRSSNFTDNAKNIIVQLKESVEINCTRPNNNTRKSIHI GPGRAFYTTGD11GD1RQAHCN1SRTKWNNTLNQ1ATKLKEQFGNNKT1VFNQSSGGDPE1VM HSFNCGGEFFYCNSTQLFNSTWNFNGTWNLTQSNGTEGNDTITLPCRIKQIINMWQEVGKA MYAPPIRGQIRCSSNITGLILTRDGGNNHNNDTETFRPGGGDMRDNWRSELYKYKVVKIEPL GVAPTKAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGVASMTLTVQARQLLSGIVQQQ NNLLRAIEAQQHLLKLTVWGIKQLQARVLTVERYLRDQQLLGIWGCSGKLICTTAVPWNAS WSNKTLDMIWNNMTWMEWEREIDNYTGLIYTLIEESQNQQEKNEQELLELDKWASLWNW FDITNWLWYIKIFIMIVGGLIGLRIVFTVLSIVNRVRQGYSPLSFQTHLPAPRGPDRPEGIEEEG GDRDRDRSVRLVDGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 4; i.e., Env protomer sequence from HIV-1 strain HIV-1AD8)[00741 In embodiments, modified SEQ ID NO: 4 comprises a variation at amino acid position 59 from lysine to alanine (i.e., K59A) and a variation at amino acid position 574 from lysine to arginine (i.e., K574R); a variation at amino acid position 136 from asparagine to glutamic acid (i.e., N136E) and a variation at amino acid position 574 from lysine to arginine (i.e., K574R); or SEQ ID NO: 4 comprises a variation at amino acid position 59 from lysine to alanine (i.e., K59A), a variation at amino acid position 136 from asparagine to glutamic acid (i.e., N136E), and a variation at amino acid position 574 from lysine to arginine (i.e., K574R). In embodiments, the immunogenic compositions herein comprise a nucleic acid sequence encoding modified SEQ ID NO: 4. All envelope glycoprotein residues in our Env mutants are numbered according to standard convention in the field (e.g., Korber BT, Foley BT, Kuiken CL, Pillai SK and Sodroski JG. 1998. Numbering positions in HIV relative to HXB2cg. HIV Sequence Compendium 1998. Los Alamos National Laboratory. Theoretical Biology and Biophysics, Los Alamos, New Mexico).

[0075] In embodiments, the protomer binds with two additional protomers and forms an Env trimer. In embodiments, one, two, or all three protomers comprise modified SEQ ID NO: 5: RVRGIPRNWPQWWIWGILGFWMIIICRVVGNLDLWVTVYYGVPVWKEAKTTLFCASDAKA YDKEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDIISLWDESLKPC VKLTPLCVTLNCKNVEISANANATATLNSSMNGEIKNCSFNTTTELRDKKQKVYALFYKPD VVPLNGGEHNETGEYILINCNSSTITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPC NNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIVRSENLTNNIKTIIVHLNKSVEIKCTRPNNNT RKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQVKEKLREHYNKTIKFEPSSGGDLE VTTHSFNCRGEFFYCDTTKLFNETKLFNESEYVDNKTIILPCRIKQIINMWQEVGRAMYAPPI EGNITCKSNITGLLLTWDGGENSTEGVFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTKS KRKVVGREKRAVGLGAVLLGFLGAAGSTMGVASMTLTVQARQLLSG1VQQQSNLLRA1EA QQHLLKLT VWGIRQLQTRVLtlERYLKDQQLLGLWGCS GKLIC ATA VPWNS S WSNKSLGDI WDNMTWMQWDREISNYTNTIFRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDITNWLW YIKIFIMIVGGLIGLRIIFGVLAIVKRVRQGYSPLSFQTLIPNPRGPDRLGRIEEEGGEQDKDRSI RLVSGFLALAWDDLRSLCLFSYHQLRDFILTAARAAELLGRSSLRGLQRGWEVLKYLGNLV QYWGLELKRSAINLFDTIAIAVAEGTDRIIEVIQRICRAIRYIPTRIRQGFEAALL (SEQ ID NO: 5; i.e., Env protomer sequence from HIV-1 strain HIV-lDu422.1)

[0076] In embodiments, modified SEQ ID NO: 5 comprises a variation at amino acid position 59 from lysine to alanine (i.e., K59A) and a variation at amino acid position 574 from lysine to arginine (i.e., K574R); a variation at amino acid position 135 from asparagine to glutamic acid (i.e., N135E) and a variation at amino acid position 574 from lysine to arginine (i.e., K574R); or SEQ ID NO: 5 comprises a variation at amino acid position 59 from lysine to alanine (i.e., K59A), a variation at amino acid position 135 from asparagine to glutamic acid (i.e., N135E), and a variation at amino acid position 574 from lysine to arginine (i.e., K574R). In embodiments, the immunogenic compositions herein comprise a nucleic acid sequence encoding modified SEQ ID NO: 5. All envelope glycoprotein residues in our Env mutants are numbered according to standard convention in the field (e.g., Korber BT, Foley BT, Kuiken CL, Pillai SK and Sodroski JG. 1998. Numbering positions in HIV relative to HXB2cg. HIV Sequence Compendium 1998. Los Alamos National Laboratory. Theoretical Biology and Biophysics, Los Alamos, New Mexico).

[0077] In embodiments, the protomer binds with two additional protomers and forms an Env trimer. In embodiments, one, two, or all three protomers comprise modified SEQ ID NO: 6: MRVKEMKRNWWGWGILLLGLLMTCSVTGKLWVTVYYGVPVWKEANATLFCASDAKAY KAEAHNIWATHACVPTDPNPQEVILENVTENFNMWKNNMVEQMHEDIISLWDESLKPCVK LTPLCVTLNCTDNVNVINATGTEISSNSTGMTNCSFNMTTELKDKQKKVTSLFYKLDVVPID TENNNNSSYNSYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGKGPCTNV STVQCTHGIKPVVSTQLLLNGSLAEEEIMIRSENLTDNAKTIIVQLNKSIAINCTRPSNNTRQS TRIGPGQVFYRTTDIIGDIRQAHCNISKKQWNETLEQVAEKLGDLFKKTTIIFKPSSGGDPEIT THSFNCGGEFFYCNTSRLFNSTWTKNNTWANNSTINETEVITLPCRIKQIINMWQGVGKAM YAPPIAGKIHCSSNITGLLLTRDGGSPNNGTNDTFRPGGGDMRDNWRSELYKYKVVRIEPLG LAPTKAKRRVVEREKRAIGLGALFLGFLGTAGSTMGVASMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLKLTVWGIKQLQARVLTVERYLKDQQLLGIWGCSGKHICTTNVPWNSSWSN RSLDY1WNNMTWMEWEKE1DNYTGV1YSL1EESQLQQERNEKDLLELDKWASLWNWFA1S NWLWYIKIFIMIVGGLIGLRIVFAVLSIVNRVRQGYSPLSFQTLLPTPRGPARPEGIEEEGGEQ GRDRSIRLLTGLSELIWDDLRNLCLFSYHHLRDLILIAARIVQLLGRRGWEALKYLWNILQY WIQELKNSAISLFDTIAIAVAGGTDRIIELAQRLGRGILNIPTRIRQGLERALL (SEQ ID NO: 6; i.e., Env protomer sequence from HIV-1 strain HIV-1191859)

[0078] In embodiments, modified SEQ ID NO: 6 comprises a variation at amino acid position 59 from lysine to alanine (i.e., K59A) and a variation at amino acid position 574 from lysine to arginine (i.e., K574R); a variation at amino acid position 139 from asparagine to glutamic acid (i.e., N139E)and a variation at amino acid position 574 from lysine to arginine (i.e., K574R); or SEQ ID NO: 6 comprises a variation at amino acid position 59 from lysine to alanine (i.e., K59A), a variation at amino acid position 139 from asparagine to glutamic acid (i.e., N139E), and a variation at amino acid position 574 from lysine to arginine (i.e., K574R). In embodiments, the immunogenic compositions herein comprise a nucleic acid sequence encoding modified SEQ ID NO: 6. All envelope glycoprotein residues in our Env mutants are numbered according to standard convention in the field (e.g., Korber BT, Foley BT, Kuiken CL, Pillai SK and Sodroski JG. 1998. Numbering positions in HIV relative to HXB2cg. HIV Sequence Compendium 1998. Los Alamos National Laboratory. Theoretical Biology and Biophysics, Los Alamos, New Mexico).

[0079] Other HIV - 1 strains : In embodiments, the changes that stabilize the HIV-1AD8, HIV- 1DU422.1 and HIV-1191859 Env trimers can also be applied to HIV-1 Envs from other strains of HIV-1. These changes include a change in residue 114 to Glu, 567 to Lys, 582 to Thr, 532 to Vai, 535 to Met, 543 to Gin, 59 to Ala, 574 to Arg, 136 to Glu (or equivalent changes that remove the VI glycan near this position), or some combinations thereof in which most of these changes are included. In particular embodiments, HIV-1 Env variant combinations include the following changes: 114E, 567K, 582T, 532V, 535M, 543Q and 574R. (E.g., Korber BT, Foley BT, Kuiken CL, Pillai SK and Sodroski JG. 1998. Numbering positions in HIV relative to HXB2cg. HIV Sequence Compendium 1998. Los Alamos National Laboratory. Theoretical Biology and Biophysics, Los Alamos, New Mexico).METHODS

[0080] The present disclosure provides methods of treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an immunogenic composition or vaccine of the present disclosure. Particularly, the present disclosure provides a method of treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure. Particularly, the present disclosure provides a method of treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of a vaccine of the present disclosure. Particularly, the present disclosure provides a method of treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure to invoke a prophylactic immunogenic response.Particularly, the present disclosure provides a method of treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of a vaccine of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a method of treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure to invoke a therapeutic immunogenic response. Particularly, the present disclosure provides a method of treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1 ) an effective amount of a vaccine of the present disclosure to invoke a therapeutic immunogenic response. In embodiments, the patient or subject in need thereof is a mammal. In embodiments, the patient or subject in need thereof is a human.

[0081] The present disclosure provides uses of an immunogenic composition or a vaccine of the present disclosure for treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an immunogenic composition or vaccine of the present disclosure. Particularly, the present disclosure provides uses of an immunogenic composition of the present disclosure for treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure. Particularly, the present disclosure provides uses of a vaccine of the present disclosure for treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of a vaccine of the present disclosure. Particularly, the present disclosure provides uses of an immunogenic composition of the present disclosure for treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides uses of a vaccine of the present disclosure for treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of a vaccine of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides uses of an immunogenic composition of the present disclosure for treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure to invoke a therapeutic immunogenic response. Particularly, the present disclosure provides uses of a vaccine of the presentdisclosure for treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of a vaccine of the present disclosure to invoke a therapeutic immunogenic response. In embodiments, the patient or subject in need thereof is a mammal. In embodiments, the patient or subject in need thereof is a human.

[0082] The present disclosure provides an immunogenic composition or a vaccine of the present disclosure for use in treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an immunogenic composition or vaccine of the present disclosure. Particularly, the present disclosure provides an immunogenic composition of the present disclosure for use in treating HIV-1 , comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure. Particularly, the present disclosure provides a vaccine of the present disclosure for use in treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of a vaccine of the present disclosure. Particularly, the present disclosure provides an immunogenic composition of the present disclosure for use in treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of an immunogenic composition of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a vaccine of the present disclosure for use in treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV-1) an effective amount of a vaccine of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides an immunogenic composition of the present disclosure for use in treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV- 1) an effective amount of an immunogenic composition of the present disclosure to invoke a therapeutic immunogenic response. Particularly, the present disclosure provides a vaccine of the present disclosure for use in treating HIV-1, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of HIV- 1) an effective amount of a vaccine of the present disclosure to invoke a therapeutic immunogenic response. In embodiments, the patient or subject in need thereof is a mammal. In embodiments, the patient or subject in need thereof is a human.

[0083] The present disclosure provides methods of invoking an immune response, comprising administering to a subject an immunogenic composition or vaccine of the present disclosure. Particularly, the present disclosure provides a method of invoking an immune response, comprisingadministering to a subject an effective amount of an immunogenic composition of the present disclosure. Particularly, the present disclosure provides a method of invoking an immune response, comprising administering to a subject an effective amount of a vaccine of the present disclosure. Particularly, the present disclosure provides a method of invoking an immune response, comprising administering to a subject an effective amount of an immunogenic composition of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a method of invoking an immune response, comprising administering to a subject an effective amount of a vaccine of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a method of invoking an immune response, comprising administering to a subject an effective amount of an immunogenic composition of the present disclosure to invoke a therapeutic immunogenic response. Particularly, the present disclosure provides a method of invoking an immune response, comprising administering to a subject an effective amount of a vaccine of the present disclosure to invoke a therapeutic immunogenic response. In embodiments, the subject in need thereof is a mammal. In embodiments, the subject in need thereof is a human.

[0084] The present disclosure provides methods of generating antibodies, comprising administering to a subject an immunogenic composition or vaccine of the present disclosure. In embodiments, the antibodies are broadly neutralizing antibodies. Particularly, the present disclosure provides a method of generating antibodies, comprising administering to a subject an effective amount of an immunogenic composition of the present disclosure. Particularly, the present disclosure provides a method of generating antibodies, comprising administering to a subject an effective amount of a vaccine of the present disclosure. Particularly, the present disclosure provides a method of generating broadly neutralizing antibodies, comprising administering to a subject an effective amount of an immunogenic composition of the present disclosure. Particularly, the present disclosure provides a method of generating broadly neutralizing antibodies, comprising administering to a subject an effective amount of a vaccine of the present disclosure. Particularly, the present disclosure provides a method of generating antibodies, comprising administering to a subject an effective amount of an immunogenic composition of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a method of generating antibodies, comprising administering to a subject an effective amount of a vaccine of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a method of generating broadly neutralizing antibodies, comprisingadministering to a subject an effective amount of an immunogenic composition of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a method of generating broadly neutralizing antibodies, comprising administering to a subject an effective amount of a vaccine of the present disclosure to invoke a prophylactic immunogenic response. Particularly, the present disclosure provides a method of generating antibodies, comprising administering to a subject an effective amount of an immunogenic composition of the present disclosure to invoke a therapeutic immunogenic response. Particularly, the present disclosure provides a method of generating antibodies, comprising administering to a subject an effective amount of a vaccine of the present disclosure to invoke a therapeutic immunogenic response. Particularly, the present disclosure provides a method of generating broadly neutralizing antibodies, comprising administering to a subject an effective amount of an immunogenic composition of the present disclosure to invoke a therapeutic immunogenic response. Particularly, the present disclosure provides a method of generating broadly neutralizing antibodies, comprising administering to a subject an effective amount of a vaccine of the present disclosure to invoke a therapeutic immunogenic response. In embodiments, the subject in need thereof is a mammal. In embodiments, the subject in need thereof is a human.

[0085] The present disclosure also provides methods of producing an immunogenic composition or vaccine as described herein. In some embodiments, the method of producing an immunogenic composition includes production of virus particles comprising a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer as described herein.

[0086] In some embodiments, the methods of producing virus particles comprising a modified human immunodeficiency virus- 1 (HIV-l) envelope glycoprotein (Env) trimer as described herein comprises stabilizing a cell line for inducible expression. In some embodiments, A549 cells are used. In some embodiments, the expression is induced using tetracycline. In some embodiments, the viral particles are replication-defective. In embodiments, the method of producing immunogenic compositions comprises modifying proviruses for inclusion of stop codons in the open reading frames for pol, reverse transcriptase, RNase H and / or integrase.TERMS

[0087] "Immunogen" refers to a substance that induces a specific immune response in a host animal (e.g., a host mammal or human). The immunogen may comprise a whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insertwith immunogenic properties; a piece or fragment of DNA or other nucleic acid capable of inducing an immune response upon presentation to a host animal (e.g., a host mammal or human); a protein, a polypeptide, a peptide, an epitope, a hapten, or any combination thereof. An immunogen generally encompasses any immunogenic substance, i.e., any substance that elicits an immune response (e.g., the production of specific antibody molecules, such as broadly neutralizing antibodies) when introduced into the tissues of a susceptible animal (e.g., a host mammal or human), and that is capable of binding to an antibody that is produced in response to the introduction of the immunogen. An immunogen is capable of being recognized by the immune system, inducing a humoral immune response, and / or inducing a cellular immune response leading to the activation of B-and / or T- lymphocytes. An immunogen may include a single epitope, or two or more epitopes. Immunogens are also referred to as antigens.

[0088] Used herein, an "antibody" is an immunoglobulin molecule comprising 2 HCs and 2 LCs interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the CDRs contained therein. The CDRs are interspersed with regions that are more conserved, termed framework regions ("FR"). An antibody is “broadly neutralizing” if it is capable of neutralizing more than one variant of a virus (e.g., HIV-1). Used herein, a “neutralizing” antibody is one that can block the entry of a pathogen into a cell so that it is unable to infect healthy cells and is unable to replicate and cause severe infection.

[0089] Used herein, an "immunogenic composition" is a composition that, when introduced into a subject (such as a mammal or a human subject), reacts with the subject's immune system molecules, i.e., induces an immune response in the subject. Immunogenic composition of the present disclosure may be provided as a recombinant protein, a purified subunit, a viral vector expressing the protein, or may be provided in the form of an inactivated vims vaccine, e.g., a live- attenuated virus vaccine, a heat-killed virus vaccine, etc.

[0090] Contemplated herein are conservative variants of the disclosed amino acid sequences herein. A protein (e.g., a modified HIV-1 Env protomer) is a conservative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein. For example, a a modified HIV-1 Env protomer that forms an Env trimer can include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, and form the Env trimer. Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art. The following groups are examples of amino acids that are consideredconservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0091] Herein, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as a modified HIV-1 Env protomer) that includes a sequence that is degenerate based on the genetic code (i.e., the 20 natural amino acids can be specified by more than one codon). All degenerate nucleotide sequences encoding the disclosed antibody and fragment polypeptide sequences are included.

[0092] Used herein, an "adjuvant" means one or more substances that enhance the immunogenicity or efficacy of an immunogenic composition or a vaccine. Some non-limiting examples of adjuvants include but are not limited to, alhydrogel, poly (EC), CpG oligodeoxynucleotide (ODN), monophosphoryl lipid A (MPLA) in liposomes, Quil-A and squalene.

[0093] Used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) or such like, or a combination thereof that is pharmaceutically acceptable for administration to the subject (e.g., mammal or human subject). The use of one or more delivery vehicles for chemical compounds in general, and immunogens in particular, is well-known to those of ordinary skill in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the diagnostic, prophylactic, and therapeutic compositions is contemplated. One or more supplementary active ingredient(s) may also be incorporated into, or administered in association with, one or more of the disclosed immunogenic compositions and vaccine formulations thereof.

[0094] Herein, an “effective amount” is a quantity sufficient to achieve a desired effect in a subject (e.g., a mammal or human subject or patient). For instance, this can be the amount necessary to prevent, treat, or ameliorate a disease, for example, inhibiting or suppressing HIV-1. In embodiments, an effective amount is the amount necessary to eliminate, reduce the infection, or prevent infection of HIV-1. Efficacy is first evident in the latent reservoir (LR) analyses, for which various assays are available, such as viral outgrowth assay or PCR-based assays. Thomas J, Ruggiero A, Paxton WA, and Pollakis G (2020) Measuring the Success of HIV-1 Cure Strategies. Front. Cell. Infect. Microbiol. 10:134. doi: 10.3389 / fcimb.2020.00134. In embodiments, an effective amount is the amount necessary to significantly inhibit or reduce HIV-1 symptoms or infection. A cellular response manifests as significantly symptoms, reduced or inhibited infection, and improvement in survival in a subject or patient. More particularly, an effective amount provides improvement in important HIV-1 endpoints, such as clinical endpoints or virologic endpoints. SeeDept, of Health and Human Services, Food and Drug Admin, Human Immunodeficiency Virus- 1 Infection: Developing Antiretroviral Drugs for Treatment Guidance for Industry (Revision 1, 2015).

[0095] HIV-1 is the most common type of HIV (Human Immunodeficiency Virus). The virus attacks the human body's immune system by destroying CD4 cells, which help the body fight infections. These attacks can lead to AIDS (Acquired Immune Deficiency Syndrome). HIV-1 has long been the subject of substantial research efforts in the scientific community. However, traditional means of generating treatments for viruses are thwarted, in part, by the structural lability and many and varied post-translational modifications of the limited surface-exposed proteins of HIV-1 (Env protein). Particularly, a State- 1 stabilized surface-exposed Env protein of HIV- 1 is the conformation that binds CD4 on the target host immune cells. Therefore, inhibitors, such as broadly neutralizing antibodies and small molecule target (CD4) mimetics have been targeted to the State- 1 stabilized conformation, and such HIV-1 Env proteins that are stabilized in the State- 1 conformation are determined empirically, given the varied and confounding factors contributing to HIV-1 Env protein State-1 stability. Provided herein are State-1 stabilized HIV-1 Env protein, which can be used to invoke an immunogenic response, such as broadly neutralizing antibodies, given that most HIV-1 in the human body exists in this State- 1, pretriggered conformation.

[0096] As used herein, the terms "immunize" or "immunization" or similar terms refer to conferring the ability to mount a detectable, or preferably, a substantial immune response against a specific antigenic epitope or immunogen. These terms do not necessarily infer complete immunity, but rather that an immune response be produced that is substantially greater than baseline, e.g., where immunogenic compositions of the invention are not administered or where a conventional vaccine is administered. For example, a mammal is considered to be immunized against one or more target immunogens, if a cellular and / or humoral immune response to the target immunogen(s) occurs (and preferably a substantial immune response) following administration of the immunogenic compositions or vaccines disclosed herein.

[0097] As used herein, the term "immunogenic response" to a composition or vaccine denotes the development of a cellular and / or antibody -mediated immune response in the host animal (e.g., mammal or human). Generally, an immunogenic response includes (but is not restricted to) one or more of the following effects: (a) the production of antibodies (e.g., broadly neutralizing antibodies); (b) the production of B cells; (c) the production of helper T cells; and / or (d) the production of cytotoxic T cells, that are specifically directed to a given antigen or hapten.

[0098] As used herein, the term "immunogenic" as used herein also refers to a substance, such as an amino acid sequence, a portion of an amino acid sequence within a protein, polypeptide, or peptide, or a modified or attenuated killed or live virus that elicits an immunological response in a host animal (e.g., mammal or human). As used herein, the term "immunogenic protein," "immunogenic peptide," or "immunogenic polypeptide" refers to proteins, peptides, and polypeptides that are immunologically active in the sense that once administered to the host (e.g., mammal or human), it is able to evoke an immune response of the humoral and / or cellular type directed against the protein. Immunogenic compositions can further contain or encode an immunogenic substance, such as a nucleic acid sequence encoding an amino acid sequence of an immunogenic polypeptide, such as a protomer (e.g., an HIV-1 protomer that forms a trimer).

[0099] Used herein, the term "individual" (also interchangeably referred to as "host”, "subject”, "recipient", "patient," etc.) refers to any animal that can receive one or more of the pharmaceutical compositions or vaccine formulations disclosed herein. Preferably, the subject is a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species, such as a human). In certain embodiments, the individual is preferably any mammalian host, including but not limited to human subjects or patients.

[0100] The pharmaceutically acceptable carriers of use are conventional (e.g., as described in Remington, The Science and Practice of Pharmacy, 22nd Edition, Loyd V., ed., Pharmaceutical Press, 2012). In general, the nature of the carrier will depend on the mode of administration. For instance, parenteral formulations typically comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions or the like as a vehicle. Pharmaceutical compositions can additionally include minor amounts of nontoxic auxiliary substances for stability.

[0101] In embodiments, the carrier may be sterile and / or suspended or otherwise contained in a unit dosage form including one or more measured doses of the composition suitable for administration to a subject of an effective amount of immunogenic compositions or vaccines disclosed herein. Medications for use in treatment may also be included in embodiments. In embodiments, the unit dosage form may be in a sealed vial that contains sterile contents or a syringe for injection into a subject, lyophilized for subsequent solubilization and administration, or in a solid or controlled release dosage.

[0102] A pharmaceutical composition of the present disclosure contains an "effective" or "therapeutically effective" amount, as used interchangeably herein, of an immunogenic compositionor vaccine of the present disclosure. The dosages and dosage regimen to achieve the desired therapeutic result depending on the means of administration and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the immunogenic composition or vaccine to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the immunogenic composition or vaccine of the present disclosure are outweighed by the therapeutically beneficial effects.

[0103] The term “therapeutic” in conjunction immunogenic compositions or vaccines disclosed herein refers to immunogenic compositions or vaccines suitable for use in human treatment of HIV- 1 . Such immunogenic compositions or vaccines are more sensitive to broadly neutralizing antibodies than the parental HIV-1 Envs from which they are derived. Any toxic or detrimental effects of the immunogenic compositions or vaccines disclosed herein are outweighed by the therapeutic or prophylactic beneficial effects.

[0104] The immunogenic compositions or vaccines disclosed herein can be used in therapy. In embodiments, the immunogenic compositions or vaccines disclosed herein can be used to treat, prevent (such as through prophylactic treatment), or ameliorate HIV-1. Herein, "preventing" a disease refers to inhibiting the full development of a disease, such as HIV-1. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in symptoms or HIV-1 infection. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as those induced by HIV-1. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology, such as AIDS. Prophylactic vaccines may be administered to an uninfected individual to prevent acquisition of HIV- 1 infection.

[0105] Used herein, the term "vaccine" refers to a composition or formulation that contains an immunogenic composition of the present disclosure in a form that is capable of being administered to a vertebrate, and preferably to an animal such as a mammal or a human. Typically, vaccines of the present disclosure will include one or more of the immunogenic compositions (including one or more modified HIV-1 Env trimers or protomers or nucleic acid sequences encoding modified HIV-1 Env protomers) disclosed herein, formulated for administration to an animal (e.g., human) in need thereof. Such compositions may be of any suitable formulation, including, without limitation, those prepared in an aqueous vehicle, as well as those in frozen, freeze-dried, lyophilized, or dehydrated form that are then subsequently rehydrated or suspended in a conventional pharmaceuticallyacceptable vehicle (e.g., sterile saline or a similar buffered aqueous solution) prior to administration. In such forms, the vaccine compositions of the present invention can be manufactured in convenient single or multiple-dose aliquots that may readily be employed in one or more of the methods or vaccination regimens disclosed herein to prevent, manage or otherwise treat one or more conditions or one or more symptoms of viral and / or microbial infection in a susceptible animal (e.g., human).

[0106] Upon introduction into the animal host (e.g., mammal or human subject or patient), the immunogenic compositions and vaccines comprising them are able to provoke an immune response, and preferably an immune response that is specific to the introduced immunogen, such that the resulting immune response is readily detectable using conventional assays known to those of ordinary skill in the immunological arts, including, but not limited to, assays detecting the production of specific antibodies (particularly broadly neutralizing antibodies that inhibit more than a small subset of HIV- 1 strains), cytokines and / or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and / or other cellular responses within the cells and tissues of the vaccinated animal. The immunogenic or vaccine compositions of the present invention may include, or be concomitantly administered in or with, one or more adjuvants alone, or in combination with one or more additional antigen(s) or such like.

[0107] The vaccines and immunogenic compositions of the present invention confer an immune response to an animal (e.g., mammal or human subject or patient) after immunization. As used herein, the term "immune response" refers to a humoral immune response and / or cellular immune response leading to the activation or proliferation of B-and / or T-lymphocytes. In some instances, however, the immune responses may be of low intensity and become detectable only when using at least one substance in accordance with the invention, while others may require repeated administration, an adjuvant, a second or further active agent, or one or more combinations thereof. The term "adjuvant" refers to an agent used to stimulate the immune system of a living organism, so that one or more functions of the immune system are increased and directed towards the immunogenic agent.

[0108] “Vector”, as used herein, is an entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and / or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct.Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some embodiments, a viral vector comprises a nucleic acid molecule encoding a disclosed modified HIV-1 Env protomer.EXAMPLES

[0109] Provided in the examples herein are combinations of Env valiants that are stabilized in a State- 1 conformation well beyond that of highly neutralization-resistant natural HIV-1 strains; the changes introduced into these Envs from multiple diverse strains of HIV- 1 enhance State- 1- associated phenotypes. Increased State- 1 stabilization correlated with increased trimer subunit association, increased sensitivity to neutralization by conformation-dependent broadly neutralizing antibodies (bNAbs), and decreased capacity to mediate membrane fusion and vims entry. Functional intermediates of the most stabilized Env mutants exhibited increased sensitivity to neutralization by pNAbs directed against the gpl20 V3 region and bNAbs against the gp41 membrane-proximal external region (MPER) after engagement of the target cell by the virus.

[0110] Example 1: Phenotypes of State- 1 -stabilized HIV-1 Envs. Compared with their unstabilized Env counterpails, viruses with functional HIV-1 Envs that are stabilized in a pretriggered (State- 1) conformation exhibit relative resistance to soluble CD4 (sCD4), CD4-mimetic compounds (CD4mcs), and exposure to cold (0°C). By screening natural Env polymorphisms, several were identified that conferred phenotypes associated with State- 1 stabilization on the HIV- 1AD8 Env. Also identified were two examples where combinations of individual Env changes enhanced State- 1- stabilized phenotypes in an additive manner: 1) a set of three changes (Q114E / Q567K / A582T) (herein called Tri) involving interior gpI20 and gp41 elements near the trimer axis; and 2) a set of three changes (A532V / 1535M / L543Q) in the gp41 fusion peptide- proximal region (herein called FPPR). To evaluate the combination of the Tri and FPPR modifications as well as other Env changes (K59A, H66N, N136E, N136Q, T138A, V255I, A316W, D325Q, R440A, and K574R) individually implicated in State-1 stabilization, HIV-1AD8 Envs were with multiple combinations of these changes were generated and assayed. The mutant Envs were evaluated for processing, subunit association, gpl20 association with detergent- solubilized trimers, and ability to mediate cell-cell fusion and pseudovirus infectivity (FIG. 1). The sensitivity of recombinant viruses pseudotyped with the Env variants to inhibition by CD4mcs and coldinactivation was also assessed (FIG. 2A). Envs that exhibited efficient processing and subunit association and were relatively resistant to CD4mcs and cold were assayed further. The mutant Envs in this panel were proteolytically processed at least as efficiently and, in most cases, more efficiently than the wild-type (wt) HIV-1AD8 Env (FIG. 1). The integrity of the Env trimer, reflected in the measured level of association between the gpl20 and gp41 subunits, was enhanced as the number of introduced Env modifications increased. Decreases in the ability of Env to mediate cell-cell fusion and virus infection also accompanied the increase in the number of introduced Env changes. As expected for Envs with potential decreases in triggerability, the viruses pseudotyped with the Tri FPPR and Tri FPPR variant Envs were markedly resistant to inhibition by the indane CD4mc, BNM- III- 170, and to cold inactivation at 0°C (FIG. 2A). For example, compared with the parental wildtype (wt) AD8 virus, the Tri FPPR virus was 56-fold more resistant to BNM-III-170 and 23-fold more resistant to cold inactivation). Based on these phenotypes, several of the Tri FPPR Env derivatives in FIG. 2A are strong candidates for State- 1 -stabilized Envs. Conversely, some combinations of Env changes failed to improve State- 1 -related phenotypes and were not assayed further (FIG. 8).

[0111] Using virus sensitivity to CD4mcs and cold as surrogates of Env triggerability, additional changes in the Tri FPPR Env were quantitatively evaluated. Sensitivity was measured for viruses pseudotyped with the Env variants to CJF-III-288, a recently discovered indoline CD4mc that inhibits HIV-1 infection more potently than BNM-III- 17027. The higher potency of CJF-III-288 was used to determine IC50 values for most of the Env variants (FIG. 2A). The product of the CJF-III- 288 IC50 and the half-life of virus infectivity at 0°C were used to calculate a State- 1 stability index, providing a quantitative ranking for the relative triggerability of the Env variants. Tri FPPR-K574R Env variants with one or more additional changes (K59A, N136E, or A316W) exhibited the highest State-1 stability indices. By these criteria, the State-1 stabilities of these HIV-1AD8 Env derivatives are beyond those found in the majority of Envs from diverse primary HIV-1 strains (FIG. 2B).

[0112] As a second approach to monitor HIV-1 Env triggerability, an assay was used in which the pseudotyped viruses were first exposed to a high concentration of either BNM-III-170 or CJF-III- 288 at 37 °C for one hour and then incubated on ice in the continuous presence of the CD4mc for up to 19 days. The half-life of virus infectivity was determined. For comparison with other stabilized HIV-1AD8 membrane Env trimers, the Comb G mutant (I535M / L543Q / K574R) was evaluated. In this more stringent assay, the infectivity of the wt AD8 virus was completely eliminated after the 1-h exposure to these high concentrations of BNM-III-170 or CJF-III-288 (FIGS. 2A and 9A-9D). TheComb G mutant was more stable, retaining 90% of its infectivity after a 1-h incubation with BNM- III- 170 but only 10% of its infectivity following an additional 24-h incubation on ice (FIG. 9D). Representative primary Tier-2 and Tier-3 neutralization-resistant viruses (JR-FL and PV0.4, respectively) likewise retained less than 15% of their initial infectivity after 24 hours in this assay. By comparison, after 72 hours of exposure to BNM-III-170 and 0°C, the virus with the Tri FPPR- K574R Env retained -75% of its initial infectivity. The infectivity of the Tri FPPR-K59A / K574R, Tri FPPR-N136E / K574R and Tri FPPR-K59A / N136E / K574R viruses in this assay was minimally affected after 19 days of incubation on ice. The results with CJF-III-288 were consistent with those obtained using BNM-III-170 (FIGS. 2A, 9B, and 9D). Thus, these more stringent assays provided quantitative data on the relative resistance of the Env mutants to CD4mc and cold, phenotypes strongly associated with stabilization of the pretriggered (State-1) conformation.

[0113] Compared with the wt AD8 virus, viruses pseudotyped with the Tri FPPR Env variants exhibited greatly diminished activation of infection of CD4-negative, CCR5 -expressing cells in response to the CD4mcs (FIG. 10A), consistent with their lower triggerability.

[0114] Example 2: CD4mc- and cold-induced gp!20 shedding from Envs on virus particles. First, the effects of Tri, FPPR and additional (K59A, H66N, N136E, A316W and K574R) changes on Env processing and incorporation into virus particles were assayed. Env processing and the levels of mature Env in virus particles were at least as great as those of the Trim FPPR Bam Env for most of the Tri FPPR Bam variants with additional changes (FIG. 3A, summarized in FIG. 3D). The levels of cleaved Env were slightly increased for some of the Tri FPPR Bam variants with combinations of additional changes.

[0115] Next, gpl20 shedding from virus particles in the presence of the CD4mc BNM-III-170 after incubation at room temperature for one hour or at 0°C for one or two days was assayed. Shedding of gpl20 from the Tri Bam Env was readily detected under all three conditions (FIG. 3B). The Tri FPPR Bam Env was much more resistant to gpl20 shedding under these conditions; the addition of the FPPR changes to the Tri Bam Env reduced gpl20 shedding by -5 fold following a 1- d incubation on ice (FIG. 3D, right column). Compared with the Tri FPPR Bam Env, the Tri FPPR Bam variants with individual additional changes (K59A, H66N, N136E, A316W or K574R) were slightly more resistant to gpl20 shedding, with K574R exerting the largest (-3.9-fold) effect. The three Tri FPPR Bam Envs with K574R combined with K59A, N136E, or both changes exhibited the most resistance to CD4mc+cold exposure, with 6.5-9.8-fold reductions in gpl20 shedding compared with that of the Tri FPPR Bam Env (FIGS. 3C-3D). The Tri FPPR Bam-K59A / K574R Env was -40times more resistant to gpl20 shedding in response to CD4mc+cold exposure than the Tri Bam Env, which, in turn, is significantly more resistant to CD4mc and cold than the wt AD8 Env. For this panel of progressively State- 1 -stabilized HIV-1AD8 Env mutants, CD4mc / cold-induced gpl20 shedding from the virus Env trimers correlated with inactivation of the functional Env spike (Spearman rS = 0.949, P < 0.05) (FIG. 4A).

[0116] Example 3: Relationship of Env State-1 stability to other HIV-1 phenotypes.Correlations between the stability of the pretriggered (State-1) HIV-1AD8 Env conformation and other HIV-1 phenotypes were examined. Not surprisingly, the State- 1 stability index positively correlated with the infectious half-life of the pseudoviruses exposed to a combination of CJF-III-288 and cold (rS = 0.849, P = 0.0001) (FIG. 11). Either the State-1 stability index or the CD4mc+cold half-life could be used to evaluate the relationship between State- 1 stability and other Env phenotypes with nearly equivalent results (FIG. 11). These indicators of State- 1 stability correlated inversely with the amount of gpl20 shed following exposure of virion Envs to a combination of CD4mc and 0°C (FIGS. 4B and 11). Both State-1 stability indices correlated positively with subunit association of the Env mutants but not with gpl20-trimer association (FIG. 4C). Subunit association monitors Env trimer stability in a native cell membrane environment, whereas gpl20-trimer association measures the stability of the Env trimers extracted from membranes by solubilization in detergent, a process that can disrupt State 1. The efficiencies with which the HIV-1AD8 Env mutants mediated cell-cell fusion and virus infection were negatively correlated with indicators of State- 1 stability (FIGS. 4D-4E and 11). Target cell levels of CD4 apparently influence the infectivity of this panel of HIV-1 AD8 Env mutants, based on the sensitivity of virus infection to the SIM.2 monoclonal antibody against CD4 (FIG. 4F). No significant relationship between the indicators of State- 1 stability and inhibition by maraviroc, a CCR5 inhibitor, was seen (FIGS. 11- 12). Thus, State- 1 stabilization of the HIV-1AD8 Env is accompanied by tighter association of the gpl20 and gp41 subunits, less responsiveness to triggering by CD4 and CD4mcs, and a decrease in membrane-fusing and virus entry functions.

[0117] Example 4: Antigenic profile of Envs on virus particles. The antigenicity of the HIV- 1AD8 Env variants on virus particles was assayed. To assay how FPPR changes affect Tri Bam Env antigenicity, recognition of Tri Bam and Tri FPPR Bam Envs was compared by a panel of bNAbs and pNAbs. As shown in FIG. 13A, no significant differences in bNAb or pNAb binding to these two Envs were detected. The mature (cleaved) Tri Bam and Tri FPPR Bam Envs were recognized efficiently by bNAbs but much less efficiently by pNAbs. Apparently, the mature Tri Bam Envexhibits a sufficiently high level of conformational stability so that the addition of the FPPR changes did not result in detectable improvement in the antigenic profile.

[0118] Next, the antigenicity of the Tri FPPR Bam Env was compared with Tri FPPR Bam Envs with double and triple combinations of K59A, N136E and K574R changes. These combination mutants exhibited high levels of Env processing, mature Env in virions, and resistance to CD4mcs and cold. The antigenicity assays in FIG. 13B focus on Env recognition by pNAbs, the binding of which has been previously correlated with Env triggerability. Immunoprecipitated Envs were deglycosylated with PNGase F to allow clear separation and easier quantification of the gpl60 and gp!20 bands. Compared with the parental Tri FPPR Bam Env, Tri FPPR Bam Envs containing the K574R change exhibited reduced pNAb-gpl20 binding (FIG. 13B). These mature Tri FPPR Bam Envs with additional modifications were efficiently recognized by bNAbs (FIG. 13C). Thus, the Tri FPPR Bam Env derivatives exhibit an antigenic profile consistent with a stabilized State- 1 conformation.

[0119] Example 5: Antibody neutralization of State- 1 -stabilized HIV-1AD8 Env variants.The sensitivity of pseudoviruses with the wt and State- 1 -stabilized mutant Envs to neutralization by sCD4-Ig and a panel of bNAbs and pNAbs was compared (FIG. 5). Compared with the wt AD8 virus, the Tri FPPR, Tri FPPR-K59A / K574R, and Tri FPPR-N136E / K574R viruses were resistant to sCD4-Ig and were slightly more sensitive to most bNAbs (FIG. 5A). One exception was the bl2 CD4-binding site (CD4BS) bNAb, which neutralized the wt HIV-1AD8 slightly more efficiently than the viruses with State- 1 -stabilized Envs. Among the CD4BS bNAbs, bl2 has previously been shown to exhibit a preference to bind a default intermediate conformation that is more open than the pretriggered (State- 1) conformation. All four viruses were resistant to neutralization by pNAbs directed against the gpl20 CD4BS, CD4-induced (CD4i) and V2 epitopes (FIG. 12), with the following exception. The Tri FPPR-N136E / K574R virus and, to a lesser extent, the Tri FPPR- K59A / K574R virus were partially neutralized by high concentrations of pNAbs (447-52D, 39F and 19b) directed against the gpl20 V3 region (FIG. 5B). This was unexpected, as the wt AD8 virus is resistant to neutralization by these V3 pNAbs, and State- 1 stabilization has previously been shown to decrease the spontaneous exposure of these and other pNAb epitopes. Moreover, as disclosed herein, the antigenicity of the mature Tri FPPR-N136E / K574R and Tri FPPR-K59A / K574R Envs on virus particles did not detect exposure of V3 pNAb epitopes (FIG. 13B). These observations argue against the spontaneous exposure of the gpl20 V3 loop as a consequence of the additional changes in the Tri FPPR-N136E / K574R and Tri FPPR-K59A / K574R Env mutants.

[0120] Virus neutralization assays were used to assess whether the V3 antibodies neutralized the State- 1 -stabilized HIV-1AD8 variants by binding the viral Env prior to or after virus engagement of the target cell. VRC01, a bNAb directed against the gpl20 CD4BS, and two MPER-directed bNAbs, 2F5 and 10E8.v4, were assayed.

[0121] In the assay of antibody binding to the viral Env prior to target cell engagement, the virus was incubated with antibody and pelleted. After removing the supernatant, the virus was added to target cells, and infection measured (FIG. 5C). In this assay format, only VRC01 neutralized the viruses with the wt AD8, Tri FPPR, Tri FPPR-K59A / K574R, and Tri FPPR-N136E / K574R Envs. The V3 pNAbs and MPER bNAbs did not inhibit infection of these viruses in this assay. Thus, these antibodies do not efficiently neutralize cell-free virus prior to engagement of the target cell.

[0122] In the assay of antibody neutralizing the virus after it attaches to the cell, viruses were incubated with target cells. After washing the cells, antibody was added, and infection was measured (FIG. 5D). In this assay, all four viruses were neutralized by the VRC01 and MPER bNAbs. The 2F5 and 10E8.v4 bNAbs neutralized the viruses with the Tri FPPR-K59A / K574R and Tri FPPR- N136E / K574R Envs more efficiently than the viruses with the wt AD8 and Tri FPPR Envs. The V3 pNAbs neutralized the viruses with the State- 1 -stabilized Envs (particularly the Tri FPPR- N136E / K574R and Tri FPPR-K59A / K574R Envs), but not the virus with the wt AD8 Env. The 447- 52D and 39F pNAbs neutralized the viruses with the State-1- stabilized Envs more effectively than the 19b pNAb. These results show that after engaging the target cell, State- 1- stabilized Env intermediates are both functional and accessible by certain V3-directed antibodies. This is not the case for the wt AD8 Env intermediates, which either do not contribute significantly to infection or are sterically or temporally inaccessible to pNAbs. The efficiency with which the State- 1 -stabilized Envs mediated infection, relative to that of the wt AD8 Env, was greater in this assay than in the cell-free virus neutralization assay shown in FIG. 5C. In addition, State- 1 -stabilized Envs may engage the multiple CD4 molecules required to establish steric blocks to V3 antibody binding less efficiently than the wt HIV-1AD8 Env. In summary, after virus-target cell engagement, functional intermediates formed by State- 1 -stabilized Envs expose certain V3 elements to antibodies.

[0123] Example 6: Phenotypes of other HIV-1 strains with State-l-stabilizing Env changes.The resulting phenotypes were evaluated for Env changes that stabilized the State- 1 conformation of the HIV-1AD8 Env would result in similar phenotypes in other unrelated HIV-1 strains: HIV- 1DU422.1 (clade C), HIV-1191859 (clade D) and HIV-1JR-FL (clade B). Where the wt Env of these HIV-1 strains had a State-l-stabilizing residue (e.g., Met 535 in JR-FL; Gin 543 in Du422.1 and191859), these were retained. State-1 stabilization of the HIV-1AD8 Env by the N136E and T138A changes results from the loss of a gpl20 VI glycan; the position of this glycosylation site shifts in the different HIV-1 strains, so the nearest glycan-modified Asn in each case was changed to Glu. The combined changes introduced into the 191859 and Du422.1 Envs moderately reduced their ability to mediate virus entry, whereas infectivity was severely impaired for the JR-FL Env mutants (FIG. 6). The processing and subunit association of the Env mutants from all three HIV-1 strains were as good or better than those of the respective wt Envs. As the Du422.1 and 191859 mutant viruses were competent for replication, they were tested for sensitivity to the CJF-III-288 CD4mc and cold inactivation. All the Du422.1 and 191859 mutant viruses were significantly resistant to CJF-III-288, cold, and the combination of CJF-III-288+cold. In the more stringent CJF-III-288+cold assay, the infectivities of the Du422.1 and 191859 mutants with multiple State- 1 -stabilizing Env changes exhibited the longest half-lives. The Du422.1 and 191859 mutant viruses inefficiently infected CD4-negative, CCR5-expressing cells in the presence of CJF-III-288 (FIGS. 10B-10C). No significant differences between the sensitivities of the wild-type (wt) and mutant viruses to the SIM.2 anti-CD4 antibody or maraviroc were observed (FIGS. 11-12). The Du422.1 and 191859 mutant viruses were more resistant to neutralization by sCD4-Ig than the respective wt viruses (FIG. 14). The wt Du422.1 virus was not neutralized by up to 10 pg / ml of the VRC01, PGT145, and 2F5 bNAbs. By comparison, the Du422.1 viruses with the introduced Env changes were more sensitive to neutralization by the PGT145, PGT151, PGT121, and 10E8.v4 bNAbs. The wt 191859 virus was resistant to the bNAbs tested, except for PGT145. The 191859 Tri FPPR-K59A / K574R virus was neutralized by several bNAbs, including VRC01, PGT151, 2F5, and 10E8.v4. Thus, the phenotypes of the HIV-1 Du422.1 and HIV-1191859 viruses with the introduced Env modifications recapitulated many of the State- 1 -associated properties observed for the HIV-1AD8 mutants.

[0124] As the viruses pseudotyped with the potentially State- 1 -stabilized HIVJR-FL Envs replicated poorly, the gp!20 shedding propensity and antigenicity of the Env trimers on virus particles were assayed. Virions were produced by transfecting HEK293T cells with the pNL4-3.JR- FL E168K plasmid encoding the JR-FL E168K Env and mutant derivatives; the E168K change allows V2 quaternary bNAbs (PG16 and PGT145) against the Env trimer apex to recognize the HIV- 1JR-FL Env. The Tri and Tri L543Q changes increased the percentage of cleaved JR-FL E168K Env on virus particles, whereas a slight decrease in the processing of the JR-FL E168K Tri FPPR Env was apparent (FIGS. 14A and 14C). JR-FL Env mutants with the Tri changes exhibited significantly reduced gpl20 shedding in response to BNM-III-170 (FIGS. 14B-14C). No significant difference inantigenicity was observed between the JR-FL E168K Env and JR-FL E168K Tri Env, although a small reduction in the precipitation of the JR-FL E168K Tri gp41 by some pNAbs was observed (FIG. 14D). When the virion Envs recognized by the antibodies were deglycosylated, the cleaved JR-FL E168K Envs with Tri and Tri L543Q changes exhibited reductions in pNAb binding (FIG. 14E). Apparently, the Tri and L543Q changes specify phenotypes in the HIV- UR- FL Env consistent with observations for the H1V-1AD8 Env.

[0125] Example 7: Methods and materials for Examples 1-6. HIV-1 Env mutants. The wildtype HIV-1AD8 env cloned in the pSVIIIenv expression plasmid was used as a template to construct HIV-1 AD8 Env mutants disclosed herein. The signal peptide / N-terminus (residues 1-33) and the cytoplasmic tail C-terminus (residues 751-856) of this Env are derived from the HIV-lHXBc2 Env. “Tri” indicates the Q114E / Q567K / A582T changes, and “FPPR” indicates the presence of Vai 532, Met 535 and Gin 543. Changes were introduced by using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies®). All the HIV-1AD8 Env valiants contain a His6 tag at the carboxyl terminus.

[0126] The Env variants from the clade C HIV-lDu422.1 strainl26 (GenBank DQ411854) and the clade D HIV-1191859 strainl25 (GenBank JX236672) were expressed from pcDNA3.1 plasmids. The pcDNA3.1 plasmids expressing the wild-type Env were obtained. Mutations were introduced into the env genes using a Q5 site-directed mutagenesis kit (New England BioLabs®). The presence of the desired mutations was confirmed by DNA sequencing.

[0127] A subset of the Env variants was expressed from env genes cloned into an infectious HIV-1 provirus in the pNL4-3 plasmid. For the HIV-1AD8 Env mutants expressed by proviruses, site- directed changes were introduced into the previously described pNL4-3.AD8 Bam plasmid; these AD8 Envs contain “Bam” changes (S752F / I756F) that decrease viral protease clipping of the gp41 cytoplasmic tail in virus particles. HIV- 1 JR-FL Env variants were expressed by the pNL4-3.JR-FL E168K plasmid; the E168K change in these Envs allows recognition by V2 quaternary bNAbsl28. Envs designated “Tri” or “FPPR” contain the changes described above. Env changes in the pNL4- 3.AD8 Bam and pNL4-3. JR-FL E168K plasmids were introduced by site-directed mutagenesis using the Q5 polymerase (New England BioLabs®). The mutated plasmids were transformed into One Shot® Stbl3 chemically competent E.coli (Life Technologies®) following the manufacturer’s protocol. All mutations were confirmed by DNA sequencing.

[0128] Antibodies, sCD4-Ig and small-molecule HIV-1 entry inhibitors. Poorly neutralizing antibodies (F105, 19b, 39F, 447-52D, 902090, 17b, E51 and F240) and broadly neutralizingantibodies (VRC01, VRC03, 3BNC117, bl2, PGT145, PG16, PGT151, 35022, PGT121, 2G12, 2F5, 4E10 and 10E8.v4) against the HIV-1 Env were obtained. The SIM.2 anti-CD4 antibody (ARP- 723) was obtained. The bNAbs include 2G12 against gpl20 outer-domain glycans; PGT121 against V3 glycans; VRC03, VRC01, 3BNC117 and bl2 against the gpl20 CD4-binding site (CD4BS); PG16 and PGT145 against quaternary V2 epitopes at the Env trimer apex; PGT151 and 35022 against the gp!20-gp41 interface; and 2F5, 4E10 and 10E8.v4 against the gp41 membrane-proximal external region (MPER). The pNAbs included b6, F105 against the gpl20 CD4BS141; 19b, 39F and 447-52D against the gpl20 V3 region 142- 144; 902090 against the gpl20 V2 regionl45; 17b and E51 against gp!20 CD4i epitopes 146- 148; and F240 against a Cluster I epitope on gp41149.

[0129] In sCD4-Ig, the N-terminal two domains of CD4 are fused with the Fc portion of an antibody.

[0130] The CD4-mimetic compounds BNM-III-170 and CJF-III-288 were synthesized. Maraviroc was purchased from Selleckchem®. The compounds were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and diluted to the appropriate concentration in cell culture medium.

[0131] Cell lines. HEK293T, TZM-bl, and HOS cells (ATCC®) were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 pg / mL penicillin-streptomycin. Cf2Th-CD4 / CCR5 and Cf2Th-CCR5 cells 153 were cultured in the same medium supplemented with 400 pg / ml G418 / 200 g / ml hygromycin or 400 pg / ml G418, respectively. All cell culture reagents were from Life Technologies®.

[0132] Env expression. To evaluate Env processing, subunit association and gpl20-trimer association, 3xl05HOS cells were seeded in 6-well plates. After 24 hours of incubation, they were transfected with pSVIIIenv plasmids encoding His6-tagged HIV-1AD8 Env variants (or pcDNA3.1 plasmids encoding HIV-lDu422.1 or HIV-1191859 Env variants), using the Lipofectamine® 3000 transfection reagent (Thermo Fisher Scientific®) according to the manufacturer’s instructions. The pSVIIIenv plasmids expressing the HIV-1AD8 Env variants were cotransfected with a Tat- expressing plasmid at an 8:1 ratio. Seventy-two hours after transfection, the supernatants were collected and incubated with Galanthus Nivalis Lectin (GNL)-agarose beads (Vector Laboratories®) for 1.5 h at room temperature. Beads were washed three times with lx phosphate-buffered saline (PBS) containing 0.1% NP-40 and processed for Western blotting with a goat anti-gpl20 antibody (Thermo Fisher Scientific®). The cells were lysed in PBS buffer containing 1.0% NP-40 and protease inhibitor (Roche® Diagnostic). For HIV-1AD8 Env variants, clarified cell lysates wereharvested and separated into two portions. One portion was used for the “Input” sample. The other was incubated with Ni-NTA beads at room temperature for 1.5 hours. Then the beads were washed, boiled, and the Env proteins analyzed by Western blotting. For the HIV-lDu422.1 and HIV- 1191859 Env variants, the cell lysates were clarified and analyzed by Western blotting. Western blots were developed with 1:2,500 goat anti-gpl20 antibody (Thermo Fisher Scientific®) and 1:2,500 HRP- conjugated rabbit anti-goat antibody (VWR®). The intensities of the gpl20 and gp!60 bands from unsaturated Western blots were quantified using ImageJ software. The Env processing index was calculated by dividing gpl20 by gpl60 in the Input / cell lysate samples. The subunit association index was calculated by dividing gp!20 in the Input / cell lysate samples by the gp!20 in the GNL precipitates. For the HIV-1AD8 Env variants, the gpl20-trimer association index was calculated by dividing the gpl20:gpl60 ratio in the Ni-NTA precipitates by the gpl20:gpl60 ratio in the Input samples. The processing and subunit association indices (and, for the HIV-1AD8 Env variants, the gpl20-trimer association indices) of the Env mutants were normalized to those of the corresponding wt HIV-1 Env.

[0133] Cell-cell fusion assay. For the alpha-complementation assay measuring cell-cell fusion, 2xl04COS-1 effector cells were seeded in black-and-white 96-well plates and then cotransfected with plasmids expressing a-gal, Env variants and Tat at a 1:1:0.125 ratio, using Lipofectamine® 3000 transfection reagent (Thermo Fisher Scientific®) following the manufacturer’ s protocol. At the same time, Cf2Th-CD4 / CCR5 cells target cells in 6-well plates were cotransfected with a plasmid expressing co-gal using Lipofectamine® 3000 transfection reagent. Forty-eight hours after transfection, target cells were detached and resuspended in medium. Medium was aspirated from the effector cells, and target cell suspensions in 50-pl volumes were added to the effector cells (one target-cell well provides sufficient cells for 50 effector-cell wells). Plates were spun at 500 x g for 3 min and then incubated at 37°C in 5% CO2 for 8 h. Medium was removed, and cells were lysed in Tropix® lysis buffer (Thermo Fisher Scientific®). The P-galactosidase activity in the cell lysates was measured using the Galacto-Star Reaction Buffer Diluent with Galacto-Star Substrate (Thermo Fisher Scientific®), according to the manufacturer’s instructions.

[0134] Virus infectivity. To produce pseudoviruses, HEK293T cells were cotransfected either with: 1) the Env-cxprcssing pSVIIIcnv plasmid, a Tat-cncoding plasmid and the luciferase-encoding pNL4-3.Luc.R-E- vector at a 1:1:3 pg DNA ratio; or 2) pSVIIIenv or pcDNA3.1 plasmids expressing Env, the pCMVAPl Aenv HIV-1 Gag-Pol packaging construct, and the firefly luciferaseexpressing HIV-1 vector at a 1:1:3 pg DNA ratio using polyethylenimine transfection reagent(Thermo Fisher Scientific®). The medium was replaced 6 to 8 h after transfection. The cell supernatants containing pseudoviruses were harvested 72 h later and centrifuged (3500 rpm for 5 min), aliquoted, and either used directly to measure pseudovirus infectivity or stored at -80°C until use.

[0135] To compare the infectivity of the valiants, pseudoviruses freshly prepared by procedures 1 and 2 were serially diluted in 96-well plates and incubated either with TZM-bl cells in the presence of 20 pg / ml DEAE-dextran or with Cf2Th-CD4 / CCR5 cells, respectively. After 48 h of incubation, cells were lysed, and the luciferase activity was measured using a luminometer.

[0136] Activation of vims infection by CD4mcs. Recombinant luciferase-expressing viruses pseudotyped with Env variants were incubated with CD4-negative, CCR5-expressing Cf2Th-CCR5 cells in 96-well plates. The plates were centrifuged at 600 x g for 30 min at room temperature. Medium containing serial dilutions of CFJ-III-288 was then added. Forty -eight hours later, the cells were lysed, and luciferase activity was measured.

[0137] Virus sensitivity to cold inactivation. To evaluate virus sensitivity to cold inactivation, pseudoviruses were incubated on ice (at 0°C) for different lengths of time and virus infectivity was subsequently measured on Cf2Th-CD4 / CCR5 or TZM-bl cells, as described above.

[0138] Virus inhibition / neutralization. The inhibitors to be tested (antibodies, sCD4-Ig, CD4mcs or Maraviroc) were serially diluted in triplicate wells of 96-well plates. Then, approximately 100 to 200 TCID50 (50% tissue culture infectious doses) of pseudoviruses was added and incubated at 37°C for 1 h. Subsequently, approximately 2 x 104TZM-bl cells (with 20 pg / ml DEAE-dextran in the medium) or Cf2Th-CD4 / CCR5 cells were added to each well and the mixture was incubated at 37°C / 5%CO for 48 h. Then the luciferase activity in the cells was measured, as described above. The concentrations of antibodies and other Env ligands that inhibit 50% of infection (the IC50 values) were determined by fitting the data in four-parameter dose-response curves using GraphPad® Prism 9.

[0139] In an assay designed to evaluate the ability of an antibody to neutralize viruses before they engage the target cell, recombinant viruses pseudotyped with Env variants were incubated for 1 h at 37°C with the following concentrations of antibodies: VRC01 (10 pg / ml), 2F5 and 4E10 (20 pg / ml), and 19b, 39F and 447-52D (50 pg / ml). The viruses were then pelleted, and the antibody-containing supernatants removed. The virus pellet was resuspended in antibody-free medium and incubated with TZM-bl cells. Forty-eight hours later, luciferase activity was measured.

[0140] In an assay designed to measure the ability of antibodies to neutralize virus after virus-cell interaction, recombinant viruses pseudotyped with the Env variants were added to TZM-bl cells. The virus-cell mixtures were centrifuged at 1800 x g for 30 minutes. The medium was removed and replaced with fresh medium containing the antibody. The concentrations of the antibodies were the same as those used in the virus neutralization assay above. Forty-eight hours later, luciferase activity in the TZM-bl cells was measured.

[0141] Virus sensitivity to combined exposure to a CD4mc and 0°C. To evaluate the sensitivity of viruses to inhibition by a combined exposure to a CD4mc and cold, pseudoviruses were incubated with either 100 pM BNM-III-170 or 10 pM CJF-III-288 or, as a control, the equivalent concentration of DMSO for 1 h at 37°C. The virus preparations were then incubated on ice for varying lengths of time (up to 19 days). The infectivity of the pseudovirus preparations for TZM-bl or Cf2Th-CD4 / CCR5 cells was measured as described above. The half-life of virus infectivity relative to the infectivity of the DMSO-treated virus at day 0 was calculated by fitting the data to a one-phase exponential decay model with GraphPad® Prism 9.

[0142] Analysis of Env on infectious virus particles produced from proviral clones. To produce virus particles, HEK293T cells were transfected with pNL4-3.env plasmids using polyethyleneimine (Thermo Fisher Scientific®). The medium was replaced 4 h after transfection, and the cells were incubated at 37°C in 5% CO2. Seventy-two hours after transfection, the cells were lysed and the supernatants were collected, filtered (0.45 pm) and centrifuged at 14,000 x g or 100,000 x g for 1 h at 4°C to concentrate the virus particles. The pelleted virus particles were resuspended in lx PBS.

[0143] To evaluate Env expression and incorporation into virus particles, clarified cell lysates and the virus particle suspensions were analyzed by Western blotting using a nitrocellulose membrane and wet transfer (350 A, 75 min, Bio-Rad®). Western blots were incubated with 1:2,000 goat anti- gpl20 polyclonal antibody (Invitrogen®), 1:2,000 4E10 anti-gp41 antibody, 1:1,000 mouse anti-p24 serum and 1:10,000 rabbit anti-hsp70 (K-20) antibody (Santa Cruz Biotechnology®). The respective HRP-conjugated secondary antibodies were 1:2,000 rabbit anti-goat (VWR®), 1:2,000 goat antihuman (Jackson ImmunoReseai'ch), 1:1,000 goat anti-mouse (Jackson ImmunoResearch) and 1:10,000 goat anti-rabbit (Cytiva®).

[0144] Shedding of gp!20 from virus particles. Vims particles resuspended in lx PBS were divided into 50-pL aliquots and incubated with serial dilutions of the CD4mc BNM-III-170 for 1 h at room temperature or on ice for 1-2 days. Next, 200 pL lx PBS was added, and the samples were centrifuged at 14,000 x g for 1 h at 4°C. Then, 220 pL of the supernatants was collected and rotatedduring incubation with Galanthus Nivalis Lectin (GNL)-agarose beads (Vector Laboratories®) for 2 h at room temperature. Beads were washed three times with lx PBS / 0.1% NP-40 and processed for Western blotting with a goat anti-gpl20 antibody. An aliquot of the virus particle suspension prior to incubation with BNM-III-170 was used as the “Input” sample.

[0145] Antigenicity of Env on virus particles. Fifty-pL aliquots of vims particle suspensions in lx PBS were incubated with a panel of antibodies at 10 pg / mL concentration for 1 h at room temperature. One mL of chilled lx PBS was added and samples were centrifuged at 14,000 x g for 1 h at 4°C. The pellets were lysed in 100 pL chilled lx PBS / 0.5% NP-40 / protease inhibitor cocktail. Lysates were rotated during incubation with Protein A-agarose beads for 1 h at 4°C and washed with chilled lx PBS / 0.1% NP-40 three times. The beads were resuspended in lx PBS containing NuPage® LDS Sample Buffer (Life Technologies®) and dithiothreitol (DTT) and used for Western blotting, as described above. To prepare the Input (50%) sample, half of the virus particle suspension was mixed with 1 mL chilled lx PBS and centrifuged at 14,000 x g for 1 h at 4°C; the pellet was resuspended in lx PBS / LDS / DTT.

[0146] To deglycosylate immunoprecipitated complexes, after the Protein A-agarose incubation and washing step, the virus-antibody-Protein A-agarose complexes were boiled in denaturing buffer (New England BioLabs®) for 10 min and treated with PNGase F (New England BioLabs®) for 1.5 h at 37°C according to the manufacturer’s protocol. To prepare the deglycosylated Input (50%), 25 pL of the virus particle suspension was mixed with 1 mL chilled lx PBS and centrifuged at 14,000 x g for 1 h at 4°C; the pellet was lysed in lx PBS / 0.5% NP-40 / protease inhibitor cocktail. The lysate was denatured and PNGase F-treated as described above. The treated proteins were then analyzed by reducing SDS-PAGE and Western blotting.

[0147] The intensity of protein bands on nonsaturated Western blots was quantified using the BioRad® Image Lab program. Statistical significance was evaluated by a two-tailed Student's t test.

[0148] Example 8: Conformations of Envs solubilized in Amphipol A18 lipid-nanodiscs. The identified naturally occurring HIV-1 polymorphisms that, individually and in combination, stabilized the pretriggered conformation of functional membrane Envs are characterized by strong subunit interactions and resist inactivation by soluble CD4 (sCD4-Ig), CD4-mimetic compounds, and exposure to cold (FIG. 17). Approaches to solubilize and purify these HIV-1 Envs while preserving the metastable pretriggered conformation were tested. To evaluate Env conformation, a panel of bNAbs, pNAbs, and sCD4-Ig (Example 7 and 16) were used. In the experiments depicted in Fig. 18, these Env ligands were used to precipitate three stabilized HIV-1AD8 Env variants from the surfaceof expressing cells. The Tri FPPR (TF), TF-K59A / K574R, and TF-K59A / K574R A715 Envs have six, eight, and eight stabilizing changes, respectively, compared with the parental HIV-1AD8 Env; in addition, the TF-K59A / K574R A715 Env has a deletion of the cytoplasmic tail, which increases cellsurface Env expression. The uncleaved Env variants were recognized by pNAbs as well as most bNAbs, consistent with their conformational flexibility. The cleaved TF and TF-K59A / K574R Envs were recognized by most bNAbs, with the exception of bl2, which preferentially binds Env in a conformation that is more “open” than State 1; pNAb binding to the cleaved TF and TF- K59A / K574R Envs was relatively inefficient. Thus, the observed pattern of antibody recognition of cleaved cell-surface TF and TF-K59A / K574R Envs corresponds to the sensitivity of viruses with these Envs to neutralization. The cleaved TF-K59A / K574R Env was recognized by sCD4-Ig less efficiently than the cleaved TF Envs, consistent with the higher degree of the stabilization of the former Env in a pretriggered conformation. Compared with the full-length TF-K59A / K574R Env, the TF-K59A / K574R A715 Env exhibited greater recognition by the bl2 antibody; the deletion of the cytoplasmic tail results in low-level sampling of the more open intermediate conformation preferred by the bl2 bNAb. These results corroborate that antibody recognition of cleaved cellsurface Env generally correlates with virus neutralization, whereas the conformationally flexible uncleaved Env, even when modified by stabilizing Env changes, is recognized by many pNAbs and most bNAbs. Thus, the State-1 Env conformation and the stabilizing effects of the TF and TF- K59A / K574R changes are dependent upon Env cleavage. These results provide antigenic profiles of the membrane-anchored, stabilized Envs on the cell surface that serve as standards against which the conformations of solubilized / purified Envs can be compared.

[0149] Example 9: Effects of pretriggered Env stabilization, choice of amphipathic copolymer, and BMS-806 addition on solubilized Env antigenicity. Solubilization of HIV-1 Env in Styrene-maleic acid (SMA) results in disruption of V2 quaternary bNAbs epitopes and exposure of some pNAb epitopes, indicating a loss of pretriggered Env integrity. Therefore, recently developed amphipathic copolymers, acrylic acid-co-styrene polymer (AASTY) and amphipol A18 (Table 1) were evaluated for their ability to solubilize Env in a more native conformation. The wt HIV-1AD8 Env and two Env variants with progressively stabilized pretriggered conformations, AE.2 and Tri FPPR (TF), were extracted directly from cell membranes with SMA, AASTY 11-50, and Al 8. Following Ni-NTA affinity purification, the solubilized Envs were precipitated with the 19b pNAb or the PGT145, PG9, and VRC03 bNAbs; pNAb and bNAb recognition provides an indication of non-State-1 and pretriggered conformations, respectively. The effects of adding theHIV-1 entry inhibitor BMS-806, which has been shown to stabilize a pretriggered (State- 1) conformation were also tested. The recognition of the gpl20 glycoprotein of the solubilized Envs is shown in Fig. 19. Using low 19b and high bNAb recognition as indicators, maintenance of the pretriggered Env conformation was favored by the following variables: (1) solubilization in Al 8 > AASTY 11-50 > SMA; (2) Tri FPPR > AE.2 > wt HIV-1AD8 Env; and (3) the presence of BMS- 806. Thus, the amphipathic copolymer used for Env solubilization, the stability of the pretriggered state of the solubilized Env, and the inclusion of a State- 1 -stabilizing additive all influence the representation of the pretriggered conformation in the purified Env preparations.Table 1 ; Amphipathic copolymers used

[0150] Example 10: Antigenicity profile of Envs solubilized in Amphipol A18 lipid-nanodiscs.To examine the antigenicity of Envs solubilized in Amphipol Al 8 lipid-nanodiscs in greater detail, a larger panel of antibodies recognizing different Env epitopes was used. The wt HIV-1AD8, AE.2 and Tri FPPR Envs described above were extracted from the membranes of expressing cells by A18 and purified by Ni-NTA affinity chromatography. The extraction and purification procedures were conducted in the absence and presence of BMS-806. The purified Envs were incubated with a panel of bNAbs, pNAbs, and sCD4-Ig. The precipitated Envs were analyzed by Western blotting (Fig. 20A). Consistent with their high degree of flexibility, the uncleaved Envs were recognized efficiently by most pNAbs and sCD4-Ig in both the absence and presence of BMS-806. The 2G12 bNAb and bNAbs against the CD4-binding site (VRC01, VRC03, bl2, and 3BNC117) also precipitated the uncleaved HIV-1AD8 Env precursor. The PG9 and PGT145 bNAbs against the Env trimer apex and the PGT151 and 35022 bNAbs against the gpl20-gp41 interface recognized gpl60 less efficiently than cleaved Envs. In the absence of BMS-806, the cleaved wt H1V-1AD8 Env was recognized by all bNAbs and most pNAbs, including F240. The F240 pNAb, which recognizes a gp41 Cluster I epitope, precipitated the gp41 glycoprotein from the A18-solubilized wt HIV-1AD8 Envs. F240 generally does not recognize HIV-1 Envs on the cell surface or on virus particles (see FIG. 18 for example). As gpl20 was not coprecipitated with gp41 by the F240 antibody, the precipitated gp41 likely represents “stumps” from which gpl20 has been shed. These results suggestthat the wt HIV-1AD8 Env in A18-lipid-nanodiscs samples multiple non- State-1 conformations, including those that lead to the shedding of gpl20. In the presence of BMS-806, recognition of the cleaved HIV-1AD8 Env by pNAbs and sCD4-Ig was reduced, while bNAb recognition was maintained or, for the PG9, PGT145, PGT151, and 35022 antibodies, enhanced. These observations are consistent with the stabilization of a pretriggered (State-1) Env conformation by BMS-806. In the absence of BMS-806, the A18-solubilized cleaved AE.2 and Tri FPPR Envs were recognized less efficiently by pNAbs than the cleaved wt HIV-1AD8 Env (Fig. 20), consistent with increased stability of the pretriggered conformation. Recognition of the Tri FPPR Env by the pNAbs, including F240, was lower than that of the AE.2 Env. The recognition of the AE.2 Env by pNAbs was reduced further by the addition of BMS-806. With the exception of the bl2 antibody, bNAb recognition of the A 18- solubilized cleaved AE.2 and Tri FPPR Envs was as good or better than that of the wt HIV- IAD8 Env. The results obtained with this larger panel of antibodies support stabilizing Env modifications and BMS-806 addition enrich the pretriggered Env conformation and decrease gpl20 shedding from Envs in A18-lipid nanodiscs. The antigenicity of A18-solubilized Tri FPPR (TF) Env with additional stabilizing changes (TF-K59A / K574R) and with a truncation (A715) of the gp41 cytoplasmic tail (Fig. 21) were evaluated. The Tri FPPR (TF), TF-K59A / K574R, and TF- K59A / K574R A715 Envs were extracted from the membranes of expressing cells by A18 and purified by Ni-NTA affinity chromatography. Precipitation of the purified TF and TF-K59A / K574R Envs by the antibodies and sCD4-Ig revealed a pattern of antigenicity similar to that of these Envs on the cell surface (compare Fig. 21 and 18). The recognition of the TF-K59A / K574R A715 Env by the F240 pNAb was greater than that of the other Envs. However, antibody and sCD4-Ig precipitation of all three Envs solubilized in Amphipol Al 8 lipid-nanodiscs correlated with recognition of the respective Envs expressed on the cell surface (Fig. 22). These results are consistent with the preservation of antigenically native conformations of these stabilized Envs in Al 8 lipid-nanodiscs.

[0151] Example 11: Extraction of membrane lipids by the amphipathic copolymers.Amphipathic copolymers can extract membrane lipids that are naturally in proximity to membrane proteins; the composition of these lipids can be analyzed by lipid mass spectrometry. The gp41 MPER of HIV- 1 Envs has a conserved cholesterol recognition amino acid consensus (CRAC) motif. Alterations of this motif, which potentially interact with cholesterol in the viral membrane, can affect Env function and sensitivity to neutralization by antibodies. The potential differences in the lipids associated with the Env-lipid nanoparticles prepared with SMA, AASTY 11-50, and Al 8 wereevaluated. The TF Env was extracted with SMA, AASTY 11-50, and A18 and purified by Ni-NTA affinity chromatography. Lipid mass spectrometry was used to characterize the lipid compositions of the Env-lipid nanodiscs prepared with each of the amphipathic copolymers. Three groups of lipids (sterols, sphingolipids, and glycolipids) were detected in all three Env-lipid nanoparticles. However, no consistent differences in the lipid compositions of the three Env-lipid nanoparticles were observed.

[0152] Example 12: Temperature and time dependence of A18-TF Env-lipid nanodisc stability. Some proteins like bacteriorhodopsin remain stable in Al 8 lipid-nanodiscs for more than 10 h at 50°C. The conformation of the TF Env in A 18 lipid-nanodiscs after incubation at 4°C and 37°C was examined. BMS-806 was added to all samples to improve stability. Purified TF Env in A18 was incubated at either 4°C or 37°C for 2 or 7 days, and the antigenic profile was evaluated (Fig. 22). Compared with the starting control TF Env (Day 0), recognition of bNAb epitopes moderately decreased and recognition of pNAb epitopes generally increased over time at both temperatures. These changes in epitope exposure were more pronounced after the 37°C incubation than after the 4°C incubation. At 37°C, particularly after 7 days, the cleaved Env was recognized by the bl2 bNAb and most pNAbs, including F240. Apparently, after 7 days at 37°C, the TF Env in A18 lipid-nanodiscs samples non-State-1 conformations, including trimers from which gpl20 has been shed.

[0153] Example 13: Cryoprotectants stabilize A18-solubilized HIV-1 Env during a freezethaw cycle. Freezing represents a practical method for storing proteins until they are ready to be used. HIV-1 Env is functionally inactivated by prolonged exposure to cold, a phenomenon that is thought to be due to ice crystal formation that disrupts intersubunit interactions. A select panel of antibodies (the pNAbs 19b and F240 and the bNAbs PG9 and VRC03) were used to evaluate potential changes in HIV-1 Env antigenicity after freezing and thawing the A18 lipid-nanodiscs. The purified H1V-1AD8 TF and TF-K59A / K574R Envs in A18 lipid-nanodiscs were snap-frozen in liquid nitrogen and thawed in a 37°C water bath (Fig. 24A and B, respectively). The binding of the 19b and F240 pNAbs to both Envs increased after freeze-thawing. Similar results were obtained when liquid ethane was used, which has a much higher heat conductivity than liquid nitrogen. Although liquid ethane is used to cool cryo-EM specimens rapidly and avoid ice crystal formation, the larger volumes (200 pL) of samples likely slow the average cooling rate. Cryoprotectants reduce the critical cooling rate, which represents the rate of cooling required to obtain amorphous Ad treous ice and avoid the formation of crystalline ice. A number of known cryoprotectants were tested,including sucrose, propanol, methanol, ethanol, DMSO, glycerol, and ethylene glycol, to see whether they could prevent the observed changes in Env antigenicity associated with freeze-thawing. At 10% concentrations, sucrose and glycerol outperformed the other additives in limiting the exposure of the 19b and F240 pNAb epitopes in both TF and TF-K59A / K574R Envs after freezethawing (Fig. 24).

[0154] Example 14: Comparison of the effects of freeze-thawing on membrane Env and A18- solubilized Envs. Freezing and thawing did not detectably affect the antigenic profile of the HIV- 1AD8 TFK59A / K574R Env on virus-like particles (VEPs) (Fig. 25A, upper panel). This result contrasts with the effect of freeze-thawing on the A 18-solubilized TF-K59A / K574R Env (Fig. 24B and 25A, lower panel). The Env cytoplasmic tail, outside of its natural membrane environment, may contribute to the sensitivity of the Env conformation to freezing and thawing. To examine this possibility, the full-length TF-K59A / K574R Env and the cytoplasmic tail-deleted TF-K59A / K574R A715 Env were solubilized in A18 and purified. As the TF-K59A / K574R A715 Env preparation has a high level of uncleaved gp!60, both the cytoplasmic tail-truncated and full-length Env were subjected to counterselection with the 19b and F240 pNAbs to remove gpl60. The effects of freezing and thawing on the TF-K59A / K574R A715 Env were at least as great as those on the TFK59A / K574R Env (Fig. 25 A, lower panel). Thus, the cytoplasmic tail does not increase the effect of freeze-thawing on the antigenic profile of the TF-K59A / K574R Env in A18 lipid-nanodiscs.Rather, other variables likely account for the different susceptibility of the VLP-associated and A18- solubilized Envs to the conformational disruption resulting from freezing and thawing (Fig. 25B).Example 15: Env conformation in A18-lipid nanodiscs formulated in adjuvants. The effect of several adjuvants [alhydrogel, poly (I:C), CpG oligodeoxynucleotide (ODN), monophosphoryl lipid A (MPLA) in liposomes, Quil-A and squalene] were examined on the antigenic profile of the purified TF Env in A18-lipid nanodiscs (see Table 2 for adjuvants). The purified TF Env was incubated with the different adjuvants at 37°C for 1 h, after which the binding of a small panel of antibodies (bNAbs 2G12, VRC03, PGT145, and PGT151, and pNAbs 19b and F240) was tested. The HIV-1 Env was tightly adsorbed to the alhydrogel, and therefore, the antigenicity could not be assessed. For the other adjuvants, no alteration of the antigenicity of the TF Env in A18-lipid nanodiscs was detected (Fig. 26). These results indicate that after solubilization in Al 8-lipid nanodiscs, the conformation of an Env with changes that stabilize the pretriggered state can be maintained in several types of adjuvants.

[0155] The studies herein demonstrate the contribution of three experimental variables to the maintenance of the pretriggered conformation of HIV-1 Envs extracted from the membranes of expressing cells: (1) the introduction of changes in Env that stabilize the pretriggered state; (2) the use of specific amphipathic copolymers for Env solubilization; and (3) the presence of BMS-806 (Fig. 27).Table 2: Adjuvants used

[0156] Example 16: Methods and materials for Examples 8-15.

[0157] HIV-1 Env mutants. The wt HIV- 1 AD8, AE.2, Tri FPPR, and Tri FPPR- K59A / K574R Envs were coexpressed with the Rev protein, using the natural arrangement of HIV-1 env and rev sequences. The Asp718 (Kpn I)-BamHI env fragments encoding the above Envs were inserted into the corresponding sites of the pSVIIIenv plasmid expressing the HIV-lHXBc2 Env and Rev proteins. These Envs contain a signal peptide and part of the cytoplasmic tail from the HIV-1 HXBc2 Env. The env gene encoding the cytoplasmic tail-deleted HIV-1AD8 TF-K59A / K574R A715 glycoprotein was codon-optimized and cloned into pcDNA3.1 (-). A (3G-His6) tag was added to the carboxyl terminus of all four Envs.

[0158] SMA, AASTY 11-50, and Amphipol A18. AASTY 11-50 and A18 were purchased from Cube Biotechnology. SMA (2:1) was purchased from Cray Valley and hydrolyzed as described previously (see Wang, Commun. Biol. 6:535 and Zhou, J. Virol. 97: e0032723).

[0159] Adjuvants. Aluminum hydroxide gel (Alhydrogel), poly (EC), CpG, Oligodeoxynucleotide (ODN) CpG, Quil-A Saponin, and AddaVax were purchased from InvivoGen. Monophosphoryl lipid A (MPLA) in liposomes was a kind gift from Polymun.

[0160] Reagents. BMS-378806 (herein called BMS-806) was purchased from Selleckchem. Superflow Ni-NTA was purchased from Bio-Rad.

[0161] Expression of HIV-1 Envs. Human A549 cells inducibly expressing the wt HIV-1AD8 Env, the AE.2 Env, the Tri FPPR Env, Tri FPPR-K59A / K574R Env, and Tri FPPR-K59A / K574R A715 Env were established as described previously (Zhang, J. Virol. 95: e0052921 and Zou, J. Virol. 94:e00148-20). A549 cells constitutively expressing the reverse tet transactivator (rtTA) were transduced with HIV- 1 -based lentivirus vectors expressing Rev and the Envs described above. The vector transcribes a bicistronic mRNA comprising rev and env and two selectable marker genes fused in-frame with a T2A peptide-coding sequence (puromycin-T2A-enhanced green fluorescentprotein [EGFP]). In the transduced cells, Env expression is controlled by the Tet-responsive element (TRE) promoter and tet-on transcriptional regulatory elements. Env expression was induced by treating the cells with 2 pg / mL of doxycycline. The Env-expressing cells were enriched by fluorescence-activated cell sorting for the co-expressed EGFP marker. These polyclonal A549 cell lines were used as sources of Env for the studies reported here.

[0162] Cell lines. The A549 cells inducibly expressing the wt H1V-1AD8, AE.2 Env, Tri FPPR Env, Tri FPPRK59A / K574R Env, and Tri FPPR-K59A / K574R A715 Envs were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. All cell culture reagents were purchased from Life Technologies.

[0163] Antibodies. Antibodies against HIV-1 Env were kindly supplied by Dennis Burton (Scripps), Peter Kwong and John Mascola (Vaccine Research Center, NIH), Barton Haynes (Duke), Hermann Katinger (Polymun), James Robinson (Tulane), and Marshall Posner (Mount Sinai Medical Center). In some cases, anti-Env antibodies were obtained through the NIH HIV Reagent Program. The primary antibodies for Western blotting were goat anti-gpl20 polyclonal antibody (Thermo Fisher) and the 4E10 human anti-gp41 antibody (Polymun). A horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Santa Cruz) and an HRP-conjugated goat anti-rabbit antibody (Santa Cruz) were used as secondary antibodies for Western blotting.

[0164] Immunoprecipitation of cell- surface HIV-1 Env. Doxycycline-induced A549-Env cells were washed twice with lx phosphate-buffered saline (PBS). The cells were then incubated with 10 pg / mL anti-Env antibody or soluble CD4 (sCD4-Ig) for 1 h at 4°C. After three washes in lx PBS, the cells were lysed in CA-630 lysis buffer [1% CA-630, lx PBS, lx protease inhibitor cocktail (Roche)] for 5 min on ice. The lysates were cleared by centrifugation at 13,200 x g for 10 min at 4°C, and the clarified supernatants were incubated with protein A-Sepharose beads for 1 h at room temperature. The beads were pelleted (1,000 rpm for 1 min) and washed three times with wash buffer (5x PBS, and 0.5% CA-630). The beads were suspended in 2x lithium dodecyl sulfate (LDS) sample buffer, boiled, and analyzed by Western blotting with 1:5,000 goat anti-gpl20 polyclonal antibody (Thermo Fisher) and 1:5,000 HRP-conjugated rabbit anti-goat IgG (Thermo Fisher). The HIV-1 gp41 Env was analyzed by Western blotting with the 1:5,0004E10 anti-gp41 antibody and 1:5,000 HRP-conjugated goat anti-human IgG (Santa Cruz).

[0165] For analysis of total Env expression in the cells, clarified cell lysates were prepared from cells that were induced with doxycycline but not incubated with antibodies. The clarified cell lysates were analyzed by Western blotting as described above and serve as the input samples.

[0166] Membrane purification. A549 cells expressing the wt HIV-1 AD8 or other mutant Envs were incubated with 5 mM EDTA in lx PBS at 37°C until the cells detached from the tissue culture plates. The cells were pelleted and resuspended in lx PBS. Cells were spun down at 1,500 x g for 10 min. The supernatants were removed, and homogenization buffer (10 mM Tris HC1 [pH 7.5], 250 mM sucrose, 1 mM EDTA, lx protease inhibitor cocktail) was added to the cell pellet. The cells were transferred into a glass Dounce homogenizer and homogenized with 250 strokes at room temperature. The homogenate was spun at 1,000 x g for 10 min at 4°C. The supernatants were centrifuged at 10,000 x g for 10 min at 4°C. The supernatants were spun again at 100,000 x g for 35 min at 16°C. The pellet represents the purified membrane fraction.

[0167] HIV-1 Env purification using Ni-NTA affinity chromatography. Cell membranes were prepared from A549 cells expressing HIV-1 Envs as described above. The membranes were then lysed in SMA, AASTY 11-50, or Amphipol A18 solubilization buffer [20 mM Tris-HCl (pH 8.0), 250 mM NaCl, 100 mM (NEU SCU with 0.5% SMA, AASTY 11-50, or A18] for 10 min at room temperature. The solubilized Env variants were incubated with Ni-NTA beads at room temperature for 1.5 h. After incubation, the mixture was applied to an Eco-column (Bio-Rad). The Ni-NTA beads were washed with 30 bed volumes of washing buffer [20 mM Tris-HCl [pH 8.0], 100 mM (NH4)2SO4, 1 M NaCl, 30 mM imidazole] and eluted with 10 bed volumes of elution buffer [20 mM Tris-HCl (pH 8.0), 100 mM (MT iSCri, 250 mM NaCl, 250 mM imidazole]. For the experiments where counterselection with pNAbs was employed, purified Envs were incubated with 40 pg / mL of 19b and 40 pg / mL of F240 antibodies and 200 mL of protein A-Sepharose at room temperature for 30 min. The mixture was applied to an Eco-column (Bio-Rad), and the purified Envs were collected in the flowthrough.

[0168] Antigenicity of Envs solubilized in amphipathic copolymer lipid-nanodiscs, Env s solubilized in amphipathic copolymer lipid-nanodiscs were purified with Ni-NTA beads as described above. The purified Env was aliquoted and incubated with 10 pg / mL antibodies together with Protein A-Sepharose beads for 1 h at 4°C. For the samples with BMS-806, 10 pM BMS-806 was added before cell lysis and remained in all the following steps. An aliquot without added antibody beads served as the Input sample. The precipitated proteins and Input sample were Western blotted as described above.

[0169] Lipid extraction. Lipids were extracted from 10 pL of the purified amphipathic copolymer Env-lipidnanodiscs. Lipids were isolated with a modified Bligh and Dyer protocol using dichloromethane and methanol. Briefly, 10 pL of each sample was transferred to a glass screw-captube with 90 p.L of water and incubated on ice for 10 min. Then, 2 mL of Methanol (MeOH) (Alfa Aesar- Thermo Fisher) and 0.9 mL of dichloromethane (DCM) (Sigma Aldrich) were added. After vortexing the DCM-MeOH-sample, a monophase was formed. After 30 min at room temperature, the aqueous and organic phases were separated by subsequent addition of 1 mL of water and 0.9 mL of DCM. All samples were centrifuged at 1,200 rpm for 10 min. The lower DCM organic phase was removed to a new glass tube and dried under nitrogen and a partial vacuum using a Visiprep manifold (Supelco). Lipid extracts were dissolved in lipid load solution: MeOH / DCM (50:50, vol / vol) containing 10 mM ammonium acetate (NFLAc) LC / MS grade (Fisher Chemical) for MS analysis.

[0170] Mass spectrometry. Unbiased MS / MSALL shotgun lipidomic analyses were performed by direct infusion. Approximately 100 pL of a fourfold-diluted lipid extract in lipid load solution was delivered by the PAL3 System (LEAP Technologies) into the source of a Sciex QTRAP 4000 mass spectrometer. A second isocratic pump MX Class HPLC pump (Teledyne SSI) connected to the PAL3 system was used to deliver the lipid load solution at a rate of 0.25 mL / min during all the runs. Source parameters included gases GS1 at 15 and GS2 at 20, curtain gas at 10, lonSpray voltage at 5300, temperature at 150, and collision energy at 50 eV. Each sample was injected twice, and positive and negative polarity modes were acquired simultaneously in serial experiments from a single sample infusion. Lipids were analyzed in multiple precursor ion scattering (MPIS) and the analytical quadrupole QI. The instrument was controlled and data were processed using Analyst software version 1.7.3. The acquired data were processed with LipidView version 1.3 beta and MarkerView version 1.4 (all software from Sciex SO). See Zhang, J Virol 98:e00631-24 incorporated herein by reference.

[0171] Example 17: Strategy for producing replication-defective VLPs with stabilized pretriggered Envs. Virus-like particles (VLPs) were produced using a generation of cell lines containing integrated, replication-defective, and inducible proviruses (def5 in Fig. 28A). The LTR of the def5 provirus has a modifiedU3 (U3TRE) region to render its promoter activity dependent on tetracycline induction, and a modifiedtrans-activation response (TARmod) element to dampen responsiveness to the Tat protein. These features ensure a low basal level of proviral expression in the absence of doxycycline and facilitate the derivation of stable cell lines that exhibit wild-type levels of proviral gene expression after doxycycline treatment. Multiple stop codons were introduced into the def5 provirus that eliminates the expression of reverse transcriptase (RT), RNase H, integrase (IN), Vif, and Vpr. The RT, RNase H, and integrase proteins are absolutely required forHIV-1 infection, and Vif is required for HIV-1 replication in natural target cells. VLP-producing cells were generated and transduced with the def5 provirus in two steps (Fig. 28B). First, singleround viruses were produced in packaging cells. For the packaging cells, HEK 293T cells were utilized that are Vif-permissive (Apobec3g-negative) and constitutively express the reverse tetracycline -regulated transactivator (rtTA). These HEK 293T cells were transfected transiently with a mixture of the def5 plasmid, a plasmid encoding a Vpr-RT-IN fusion protein (to complement RT, RNase H, and IN), and a plasmid expressing the VSV G glycoprotein. The transfected packaging cells were also treated with doxycycline, resulting in the production of recombinant viruses capable of a single round of infection. Second, the single-round viruses generated from the HEK 293T cells were used to stably transduce VLP producer cells. The strategy of transducing the producer cells with the def5 provirus capitalizes on the ability of HIV- 1 integration to favor active genes and local transcriptional hotspots, reducing the problem of transcriptional silencing associated with transfection-based DNA delivery. Vpu and Nef expressions by the integrated def5 provirus counteract any potential effects of Bst2 or SERINC3 / C5 in the VLP-producing cells.

[0172] Although numerous cell types can be used as VLP producer cells in this system, A549 human lung epithelial cells were used as they have been extensively studied with respect to HIV-1 Env expression and processing. To make def5 expression in the VLP-producing cells inducible by treatment with doxycycline, A549 cells constitutively expressing rtTA were established and utilized for def5 transduction.

[0173] To evaluate the effect of HIV-1 Env conformation on the phenotypes of the VLPs, the AD8 Bam Env were compared from the primary HIV-1AD8 with two HIV-1AD8 Env variants, Tri Bam and Tri FPPR Bam, which are progressively stabilized in a pretriggered conformation. Compared with the AD8 Bam Env, the Tri Bam and Tri FPPR Bam Envs have three and six changes, respectively, that stabilize the pretriggered conformation (Fig. 28). As a result, the Tri Bam and Tri FPPR Bam Envs are relatively resistant to soluble CD4 (sCD4), CD4-mimetic compounds (CD4mcs), and cold inactivation. Based on these viral phenotypes, the stability of the pretriggered Env conformation exhibits the following rank order: Tri FPPR Bam > Tri Bam > A.D.88 Bam. As their names indicate, all the Envs used herein contain Bam changes (S752F / I756F), which decrease proteolytic clipping of the gp41 cytoplasmic tail by the HIV-1 protease in viral particles. Although gp41 clipping does not detectably affect Env antigenicity, it is not evident in most HIV-1 strains and is easily avoided in the HIV-1AD8 constructs by the Bam changes. The Envs also contain an acarboxy-terminal Strep-tag II sequence to facilitate Env purification.

[0174] In pilot experiments to validate the approach, the def4 proviral construct was used instead of def5. Like the def5 provirus, def4 does not express functional RT, RNase H, IN, or Vif proteins (Fig. 28A). However, def4 has a wild-type HIV-1 LTR sequence, and therefore, unlike def5, it is not dependent on doxycycline for efficient expression. The def4 provirus is identical to the pNL4-3.env infectious provirus except for the stop codons in the pol and vif genes. The tetracycline independence of def4 eliminated the variable of doxycycline addition and made experiments to characterize the VLP Envs more convenient and interpretable, allowing direct comparisons to the infectious proviral clone pNL4-3.env. As shown below, the Env glycoproteins on VLPs produced by the def4 and def5 proviruses were indistinguishable in the assays.

[0175] Example 18: Characterization of single-round viruses produced by HEK 293T packaging cells. Env expression, processing, and incorporation into single-round viruses produced from HEK 293T packaging cells transfected with the def4 plasmid or with the corresponding infectious molecular' proviral clone pNL4-3. env was compared. In these experiments, def4 and pNL4-3.env proviruses, each encoding AD8 Bam, Tri Bam, and Tri FPPR Bam Envs were evaluated. The effectof cotransfecting the plasmid expressing the Vpr-RT-IN protein, which complements the defective RT, RNase H, and IN proteins in the def4 provirus was also examined. The stop-codon changes introduced into the def4 provirus resulted in a 2-fold reduction in virus particles and Env proteins released into the cell supernatants compared with the levels seen following pNL4-3.env transfection (Fig. 29 A); the altered ratio of Gag and Gag-Pro-Pol polyproteins in the cells expressing these two proviruses may contribute to the observed differences in released particles. However, Env processing and incorporation into virus particles were similar' for the cells expressing the def4 and pNL4-3.env proviruses, with or without Vpr-RT-IN expression. Thus, the apparent Env content of VLPs produced by the def4 and pNL4-3.env proviruses is similar.

[0176] The supernatants from the HEK 293T cells transfected with the def4 and pNL4-3.env proviral plasmids, with or without the Vpr-RT-IN expressor plasmid, were titrated on TZM-bl target cells to measure viral infectivity. In these experiments, virus entry is mediated by the HIV-1 Envs encoded by the def4 or pNL4-3.env proviruses. As expected for Envs stabilized in a pretriggered conformation, the Tri Bam and Tri FPPR Bam Envs supported virus infectivity at a lower level compared with the AD8 Env (compare the y-axis values in Fig. 29B). The Tri FPPR Bam Env, which is more stabilized in the pretriggered conformation than the Tri Bam Env, mediated virus infection less efficiently.Coexpression of Vpr-RT-IN reduced the measured infectivity of the pNL4- 3. env viruses by approximately 2-fold; presumably, the relative increase in RT, RNase H, and / or INproteins in the virions is mildly detrimental to infectivity. By contrast, Vpr-RT-IN rescued the infectivity of the def4 virus particles, which do not otherwise contain RT, RNase H, or IN. The antigenicity of the Tri FPPR Bam Env was similar to virus particles produced by transfection of the pNL4-3.env and def4 proviruses (Fig. 29C). The mature Tri FPPR Bam Env was recognized efficiently by a panel of bNAbs but not by pNAbs. The relatively small amount of the uncleaved gpl60 Env that was incorporated into VLPs was recognized by the gpl20-specific pNAbs, consistent with its conformational flexibility. The F240 pNAb, which recognizes an epitope on the gp41 ectodomain, did not precipitate the Tri FPPR Bam Env on the virus particles; thus, shedding of gp!20 from the Tri FPPR Bam Env trimers, which would expose the gp41 epitope recognized by the F240 antibody, was not detectable under these conditions. The results indicate that defective viruses expressing an Env stabilized in a pretriggered conformation can be generated by transient transfection of HEK 293T cells; one round of infection can be achieved by these viruses after complementation by the Vpr-RT-IN protein.

[0177] Example 19: Composition of VLPs produced from A549 cells stably transduced with def5. A549 cells expressing rtTA were infected with single-round def5 viruses produced transiently in transfected HEK 293T cells. The A549 cell lines were transduced at a multiplicity of infections that resulted in more than 75% of the cells staining positive for both Env and Gag expressions. The polyclonal def5-transduced A549 cell lines were used to produce VLPs for subsequent analyses. For the analysis of p24 Gag and gpl20 Env content, the polyclonal A549 cells stably transduced with def5 containing the Tri FPPR Bam Env were treated with 2 pg / mL of doxycycline. After 48 h, the cell culture supernatant was collected, clarified by centrifugation at 600 x g for 10 min, filtered through a 0.45-pm filter, precipitated by ultracentrifugation at 100,000 x g for 1 h, and then solubilized in Laemmli buffer. The VLP lysates were analyzed by western blotting and compared with purified protein standards (Fig. 30A). Quantitation of the bands detected on western blots was used to calculate the relative molar quantities of p24 Gag and gpl20 Env on the VLPs. Assuming a content of 1200 p24 Gag molecules / virion, an average VLP contains about 57 Tri FPPR Bam Env trimers (Fig. 30B and C).

[0178] The molar ratio of gpl20 to p24 Gag on VLPs was further examined by comparing VLPs that were pelleted by ultracentrifugation with or without a 20% sucrose cushion to limit the presence of extracellular vesicles. The VLP pellets were solubilized in Laemmli buffer, and the lysates were run on SDS-PAGE gels and analyzed by western blotting to quantify band intensities as described above. The western blot results show that the Gag and Env content on VLPs pelleted throughsucrose was similar to that on VLPs pelleted without a sucrose cushion (Fig. 30D). By calculating the p24 / g l20 molar ratios, the average VLP pelleted without a sucrose cushion was found to contain 35 Env trimers, whereas VLPs pelleted through 20% sucrose contained 28 Env trimers (a difference of about 20%).

[0179] To verify the incorporation of Tri FPPR Bam Env trimers on the VLPs, cryo-electron tomography (cryo-ET) was used to observe the particles produced from transduced A549 cell lines directly. The tomograms revealed high numbers of Envs on the surface of VLPs (Fig. 31 A), with an average of 25 ± 13 Envs per virion (Fig. 31B). Subtomogram averaging of -1,700 particles resulted in a 16 A Tri FPPR Bam Env structure similar to the previous low-resolution structures of Env derived from the HIV-lBaL strain (Fig. 31C). Additionally, the Tri FPPR Bam VLPs displayed heterogeneity in capsid formation, with a high proportion of particles lacking capsids (vesicles), containing immature capsids or possessing improperly sealed capsids (Fig. 31A and D). This suggests that the proviral modifications that eliminate infectivity, particularly the deletion of RT and IN, impact capsid maturation. These observations are consistent with previous studies in which abnormal HIV-1 capsid morphologies resulted from the loss of RT and IN. Although capsid maturation was affected in def5, there was no significant defect in Env incorporation among the different types of particles (Fig. 3 IE). The cryo-ET study verifies the presence of significant numbers of Tri FPPR Bam Env trimers on the surface of the VLPs produced by this approach.

[0180] Example 20: Characterization of HIV-1AD8 and stabilized pretriggered Envs on replication-defective VLPs. The Envs on VLPs were produced after doxycycline induction of the A549 cells transduced with def5. In Fig. 32A, Env processing and incorporation into vims particles prepared from HEK 293T packaging cells and A549 producer cells are compared. The efficiency of Env processing was comparable in both cell types; however, the A549 cells produced approximately twice as much mature Env (gpl20 + gp41) than the HEK 293T cells, after normalization for the number of cells (based on hsp70) and the number of particles (based on Gag p24). The VLPs produced from the A549 cells lacked detectable RT and integrase (Fig. 32B). The A549-produced VLPs were incubated with C8166-R5 T cells over an extended period, during which time the supernatant was collected and inoculated on TZM-bl cells to evaluate potential infectivity. Control viruses produced from HEK 293T cells transfected with pNL4-3.AD8 Bam efficiently infected the TZM-bl cells (Fig. 32C). As expected, a matched plasmid pNL4-3.AD8 Bam(-) with two Env changes (R508S / R511S) that eliminate gpl60 proteolytic cleavage failed to produce infectious viruses in parallel experiments. None of the def5 VLPs produced from A549 cells detectablyinfected TZM-bl cells. These results suggest that replication-defective VLPs can be inducibly produced from A549 cells. The composition and maturation of VLPs from def5-transduced A549 cells were similar after multiple passages of the cell lines (Fig. 32D).

[0181] The glycosylation and oligomeric status of Env on the A549-produced VLPs were evaluated. Env sensitivity to Endoglycosidase Hf (Endo Hf), which does not cleave complex glycans, was compared with Peptide:N-glycosidase F (PNGase F), which completely removes N- linked glycans. The gpl20 and gp41 subunits of the mature Envs were modified by complex carbohydrates resistant to Endo Hf (Fig. 32E, upper panels). A fraction of the gp41 glycoprotein was sensitive to Endo Hf digestion, indicating modification by high-mannose and / or hybrid glycans. Likewise, the small amount of gpl60 incorporated into the VLPs was partially modified by complex carbohydrates; this gpl60 apparently passed through the Golgi without being cleaved. Env glycosylation on the A549-produced VLPs resembled that of Env on virus particles from HIV-1- infected T lymphocytes (Fig. 32E, lower panels). The AD8 Bam, Tri Bam and Tri FPPR Bam Envs on the VLPs produced in A549 cells were largely trimeric (Fig. 32F).

[0182] The antigenicity of the AD8 Bam, Tri Bam, and Tri FPPR Bam Env on the VLPs produced in the A549 cells was similar to that of the Envs on virus particles derived from transiently expressing HEK293T cells (Fig. 33A). The mature AD8 Bam Env on VLPs was recognized not only by bNAbs but also by some pNAbs. Introducing the D368R change, which prevents gpl20 binding to CD4, into the AD8 Bam Env did not eliminate pNAb binding (Fig. 33B). This result argues against the interaction of Env with CD4 or a CD4-like molecule as an explanation for the exposure of pNAb epitopes. The mature Tri Bam and Tri FPPR Bam Envs were recognized less efficiently than the AD8 Bam Env by pNAbs (Fig. 33A). Relative to the AD8 Bam Env, the Tri Bam, and Tri FPPR Bam Envs on the VLPs were more resistant to the shedding of gpl20 induced by the CD4- mimetic compound, BNM-III-170 (Fig. 33C). Thus, the replication-defective VLPs produced in A549 cells contain HIV-1 Envs that retain various degrees of stability and associated antigenicity of the pretriggered Env conformation.

[0183] Example 21: Methods and materials for Examples 17-20.

[0184] Plasmids. The tetracycline-inducible, replication-defective proviruses used in this study were derived from the previously described infectious molecular clone pNL4-3.AD8 Bam (Nguyen, Virol 97:e0185722). Modifications of this proviral plasmid were performed by site-directed mutagenesis using Q5 High-Fidelity polymerase (New England Biolabs) and One Shot™ Stbl3 cells (Thermo Fisher Scientific)following the manufacturer’s protocol. A carboxy-terminal Strep-tag IIsequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) was added to the AD8 Bam Env as previously described and is retained in all Envs in this study. The def4.AD8 Bam provirus was made by the introduction of stop codons into pNL4-3.AD8 Bam sequences encoding reverse transcriptase (and RNase H and IN); integrase; integrase + Vif; and Vif. The pNL4-3.Tri Bam and pNL4-3.Tri FPPR Bam proviral plasmids encoding State- 1 -stabilized Envs have been previously reported. The Spe I- Sal 1 fragment from def4.AD8 Bam (from within gag to near the 3’ end of vpr) was amplified using Q5 high-fidelitypolymerase and inserted into the corresponding sites of pNL4-3.Tri Bam and pNL4- 3.Tri FPPR Bam plasmids. The ligation reaction was carried out using T4 ligase (New England Biolabs) at a 5:1 insert:vector molar ratio at 16°C overnight. The resulting intermediate plasmids were designated def4.Tri Bam and def4.Tri FPPR Bam, respectively. The internal sequences of the def4.AD8 Bam, def4.Tri Bam, and def4.Tri FPPR Bam proviruses were inserted into the pNL- tetOx2 proviral plasmid. During this process, a stop codon was introduced into the vpr gene to eliminate Vpr expression; the CAA codon for Gin 3 of Vpr was changed to TAA. The resulting def5 proviruses are defective in the expression of RT, Nase H, IN, Vif, and Vpr. In addition, the transactivation response (TAR) element in the def5 long terminal repeat (LTR) is mutated to reduce Tat-mediated transactivation. Two tet operator sequences have been inserted into the U3 regions of the LTRs to upregulate transcription in the presence of doxycycline (Dox). The resulting tetracycline -regulated, replication-defective proviral plasmids are designated def5.AD8 Bam, def5.Tri Bam, and def5.Tri FPPR Bam. The Kappes laboratory filenames for these plasmids are K5887 - def5.AD8 Bam; K5888 - def.5.Tri Bam; and K5889 - def5.Tri FPPR Bam.

[0185] Antibodies. Poorly neutralizing antibodies (F105, 19b, 39F, 447-52D, 17b, E51, and F240) and broadly neutralizing antibodies (VRC01, VRC03, bl2, PGT145, PG16, PGT151, 35022, PGT121, 2G12, 2F5, 4E10, and 10E8.v4) against the HIV-1 Env were obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH. The bNAbs included 2G12 against gpl20 outer-domain glycans; PGT121 against V3 glycans; VRC03, VRC01, and bl2 against the gpl20 CD4-binding site (CD4BS); PG16 and PGT145 against quaternary V2 epitopes at the Env trimer apex; PGT151 and 35022 against the gpl20-gp41 interface; and 2F5, 4E10, and 10E8.v4 against the gp41 membrane-proximal external region (MPER). The pNAbs included F105 against the gpl20 CD4BS; 19b, 39F, and 447-52D against the gpl20 V3 region; 17b and E51 against gpl20 CD4i epitopes; and F240 against a Cluster I epitope on gp41. The NIH HIV Reagent Program also provided mouse anti-reverse transcriptase monoclonal antibody clone 28, anti-p24 monoclonal antibody, and mouse anti-integrase clone 2C11.

[0186] Cell lines. HEK 293T, TZM-bl, A549 (ATCC), and rtTA-expressing HEK 293T and A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 pg / mL penicillin- streptomycin (Life Technologies). CCR5-expressing C8166-R5 T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 100 pg / mL penicillin-streptomycin; 1 pg / mL of puromycin was added at every fifthpassage.

[0187] Establishment of A549 VLP producer cells. HEK 293T packaging cells expressing the reverse tetracycline-regulated transactivator (rtTA) were transfected with def5 plasmids (def5.AD8 Bam, def5.Tri Bam, or def5.Tri FPPR Bam) along with the CMV-Vpr-RT-IN and CMV-VSV G plasmids. The medium was replaced 4 h after transfection with a fresh medium containing 2 pg / mL Dox. Cell supernatants were collected 72 h after transfection, clarifiedby low-speed centrifugation, filtered(0.45 pm), and used to infect rtTA-expressing A549 cells. The A549 VLP producer cells were maintained as polyclonal cell lines. The Kappes laboratory designations for these cell lines are: D1674 - def5.AD8 Bam; D1675 - def5.Tri Bam; and D1672 - defS.Tri FPPR Bam.

[0188] Production and preparation of viruses and VLPs. Viruses were produced by transfection of HEK 293T cells with pNL4-3.env plasmids using polyethyleneimine (PEI, Polysciences). VLPs were produced by transfection of HEK 293T cells with def4 plasmids. In some experiments, the Vpr-RT-IN expressor plasmid was cotransfected at a 1:2 (wt:wt) ratio with the pNL4-3.env and def4 plasmids. The medium was replaced 4 h after transfection; 2 pg / mL doxycycline was included in the medium of cells transfected with the def5 plasmids. At 72 h after transfection, the cells were harvested and lysed; the cell lysates were clarifiedby centrifugation at 14,000 x g for 5 min at 4°C. Viruses and VLPs were prepared from the cell supernatants collected at 72 h after transfection. VLPs prepared for analyzing Env content relative to p24 were precipitated from cell culture supernatant 48 h after treatment with doxycycline. For small-scale experiments, 1 mL supernatants were spun at 14,000 x g in microcentrifuge tubes in an Eppendorf bench-top centrifuge. For larger-scale experiments, 10 mL supernatants were spun at 100,000 x g in an ultracentrifuge. No differencein the quality of the viruses prepared by these methods was observed.

[0189] For the production of VLPs from rtTA-expressing A549 cells transduced with def5 proviruses, the cells were seeded in T75 tissue-culture flasksl6 h prior to induction. When the cells reached -80% confluency,2 pg / mL Dox was added to the medium. Approximately 72 h later, the cell medium was collected, and VLPs were prepared as described above.

[0190] Analysis of Env on virus particles and VLPs. Clarifiedcell lysates and pelleted virus particles were analyzed by western blotting using a nitrocellulose membrane and wet transfer (350 A, 75 min, Bio-Rad). Western blots were developed with 1:2,000 goat anti-gpl20 polyclonal antibody (Invitrogen), 1:2,000 4E10 anti-gp41 antibody, 1:1,000 mouse anti-p24 monoclonal antibody (AIDS Reagent Program), and 1:10,000 rabbit anti-hsp70 (K-20) antibody (Santa Cruz Biotechnology). The HRP-conjugated secondary antibodies were 1:2,000 rabbit anti-goat (Invitrogen), 1:2,000 goat anti-human (Invitrogen), 1:1,000 goat anti-mouse (Invitrogen), and 1:10,000 goat anti-rabbit (Sigma- Aldrich). The intensity of protein bands on nonsaturated western blots was quantified using the Bio-Rad Image Lab program unless otherwise noted. Statistical significance was evaluated by a two-tailed Student’s t test.

[0191] Cryo-electron tomography. Approximately 400 mL of supernatant of the A549 cell line transduced with the def5.Tri FPPR Bam provirus was collected after a 2-day induction. The supernatant was clarified by low-speed centrifugation, filtered(0.45 pm), pelleted by ultracentrifugation over a 20% sucrose cushion (130,000 x g for 2 h), and resuspended in 40 pL PBS. VLPs were mixed with 6 nm gold tracer at a 1:3 ratio, and 5 pL of the mixture was placed onto freshly glow-discharged holey carbon grids (R 2 / 1 200 mesh Cu, Quantifoil) for 1 min. Grids were blotted with filter paper, and plunge-frozen into liquid ethane by a homemade gravity-driven plunger apparatus.

[0192] Cryo-grids were imaged on a cryo-transmission electron microscope (Titan Krios, Thermo Fisher Scientific)operated at 300 kV, using a Gatan K3 direct electron detector in counting mode with a 20 eV energy slit and Volta Phase Plate (VPP). Tomographic tilt series between -60° and +60° were collected by using SerialEM in a dose-symmetric scheme with increments of 3°. The nominal magnification was 64,000x, giving a pixel size of 1.346 A on the specimen. The raw images were collected from single-axis tilt series with a cumulative dose of ~120 e per A2. The defocus was at -0.5 pm, and nine frames were saved for each tilt angle.

[0193] Frames were motion-corrected using Motioncorr2 to generate drift-corrected stack files, which were aligned using gold fiducial makers by IMOD / etomo. Tomograms were reconstructed and visualized with IMOD. All Envs were manually picked. Euler angles were determined based on the vector between Env and the center of the virion. Subtomograms were extracted for subsequent processing using 13 with 4 x binned tomograms. The averaged Tri FPPR Bam Env structure was visualized in ChimeraX.

[0194] Virus infectivity. Cell supernatants containing viruses were clarified by low-speed centrifugation (2,000 rpm for 10 min). Serial dilutions of clarified supernatants were incubated with TZM-bl cells in 96- well plates (2 x 104 cells per well). The plates were incubated at 37 °C and 5% CO2 for 48 h, after which the cells were lysed, and luciferase activity was measured using a luminometer.

[0195] Measurement of virus infectivity in C8166-R5 lymphocytes. Viruses were produced from transfected 293T cells (1 pg pNL4-3.env per well in a 12-well plate) or Dox-induced A549 cells transduced with def5 proviruses as described above. Seventy-two hours later, approximately 1 mL supernatant was filtered(0.45 pm), and 200 pL was incubated with 1 mL (2 x 106) C8166-R5 cells. Three hours later, cells were washed, 4 mL medium was added, and cells were incubated at 37 °C in 5% CO2. Every 3-5 days following infection, 3 mL of cell suspension was collected, and 3 mL of fresh medium was added. Collected cell suspensions were spun at 2,000 rpm for 10 min and clarified supernatants were stored at -80°C for analysis of infectious virus titer, as described above.Luciferase readings using 17.8 pL of virus suspensions were chosen for evaluation because the positive control AD8 Bam virus from transfected 293T cells showed maximal reading at this volume.

[0196] Deglycosylation of Env on virus particles. Purified virus particles were lysed in 1 x PBS / 0.5% NP-40. The viral lysate was then boiled in denaturing buffer(New England BioLabs) for 10 min and treated with PNGase F or Endo Hf enzymes (New England BioLabs) for 1.5 h at 37°C according to the manufacturer’s protocol. The treated proteins were analyzed by reducing SDS- PAGE and western blotting.

[0197] Analysis of the Env oligomeric state on virus particles. Purified virus particles were incubated with different concentrations of bis(sulfosuccinimidyl)suberate (BS3) crosslinker (ThermoFisher Scientific )for 30 min at room temperature, after which the reaction was quenched with 100 mM Tris-HCl, pH 8.0, for 10 min at room temperature. NuPage LDS Sample Buffer(New England Biolabs) and dithiothreitol (DTT) were added. The samples were then boiled and analyzed by reducing SDS-PAGE and western blotting.

[0198] Antigenic profile of Env on virus particles. To evaluate Env antigenicity, 50 pL aliquots of virus particles (purified and resuspended in 1 x PBS) were incubated with a panel of antibodies at 10 pg / mL concentration for 1 h at room temperature. One mL of chilled 1 x PBS was added, and samples were centrifuged at 14,000 x g for 1 h at 4°C. The pellets were lysed in 100 pL chilled 1 x PBS / 0.5% NP-40 / protease inhibitor cocktail. Lysates were rotated during incubation with protein A-agarose beads for 1 h at 4°C and washed three times with chilled 1 x PBS / 0.1% NP-40. The beads were resuspended in 1 x PBS / LDS / DTT and used for western blotting. To prepare the Input (50%) sample, half of the virus volume was mixed with 1 mL chilled 1 x PBS and centrifuged at 14,000 x g for 1 h at 4°C; the pellet was resuspended in 1 x PBS / LDS / DTT and analyzed by western blotting.

[0199] Shedding of gp!20 from virus particles. Purified virus particles were resuspended in 1 x PBS, divided into 25 pL aliquots, and incubated with serial dilutions of the CD4-mimetic compound BNM-IIL170 (195) for 1 h at room temperature. Next, 200 pL 1 x PBS was added, and samples were centrifuged at 14,000 x g for 1 h at 4°C. Then, 200 pL of the supernatants was collected and rotated during incubation with Galanthus Nivalis Lectin (GNL)-agarose beads (Vector Laboratories) for 2 h at room temperature. Beads were washed three times with 1 x PBS / 0.1% NP-40 and processed for western blotting with a goat anti-gpl20 antibody. An aliquot of the purified virus particles prior to incubation with CD4-mimetic compound was used as the “Input” sample. See Ding, J. Virol. 98:e01720-24 incorporated herein by reference.EQUIVALENTS

[0200] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

CLAIMSWhat is claimed:

1. An immunogenic composition, comprising: a. a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer, comprising: i. three modified HIV-1 Env protomers, wherein each of the three modified HIV-1 Env protomers comprise the amino acid sequence of SEQ ID NO: 1; ii. three modified HIV-1 Env protomers, wherein each of the three modifiedHIV- 1 Env protomers comprise the amino acid sequence of SEQ ID NO: 2; or iii. three modified HIV-1 Env protomers, wherein each of the three modifiedHIV-1 Env protomers comprise the amino acid sequence of SEQ ID NO: 3; or b. a nucleic acid sequence encoding a modified HIV-1 Env protomer, comprising: i. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1; ii. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; or iii. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3.

2. An immunogenic composition, comprising: a. a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) protomer sequence, comprising: i. a modified HIV-1 Env protomers comprising the amino acid sequence of:

1. SEQ ID NO: 1;2. SEQ ID NO: 2; or3. SEQ ID NO: 3; or ii. a nucleic acid sequence encoding a modified HIV-1 Env protomer, comprising:

1. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1;2. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; or3. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3; and b. a pharmaceutically acceptable carrier.

3. A vaccine, comprising: a. a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) protomer sequence, comprising: i. a modified HIV-1 Env protomer comprising the amino acid sequence of:

1. SEQ ID NO: 1;2. S EQ ID NO: 2; or3. SEQ ID NO: 3; or ii. a nucleic acid sequence encoding a modified HIV-1 Env protomer, comprising:

1. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1;2. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; or3. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3; and b. a pharmaceutically acceptable carrier.

4. The immunogenic composition of claim 1 or claim 2, wherein the modified HIV-1 Env protomer comprises SEQ ID NO: 1.

5. The immunogenic composition of claim 1 or claim 2, wherein the modified HIV-1 Env protomer comprises SEQ ID NO: 2.

6. The immunogenic composition of claim 1 or claim 2, wherein the modified HIV-1 Env protomer comprises SEQ ID NO: 3.

7. The vaccine of claim 3, wherein the modified HIV-1 Env protomer comprises SEQ ID NO:1.

8. The vaccine of claim 3, wherein the modified HIV-1 Env protomer comprises SEQ ID NO:2.

9. The vaccine of claim 3, wherein the modified HIV-1 Env protomer comprises SEQ ID NO:3.

10. The immunogenic composition of any one of claims 1-2 and 4-6, wherein the modified HIV-1 Env protomer forms a State- 1 stabilized trimer.

11. The vaccine of any one of claims 3 and 7-9, wherein the modified HIV-1 Env protomer forms a State- 1 stabilized trimer.

12. An immunogenic composition, comprising: a. a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) means for stabilizing Env trimer State- 1; and b. a pharmaceutically acceptable carrier.

13. An immunogenic composition, comprising: a. a nucleic acid sequence encoding a modified human immunodeficiency virus- 1 (HIV -1) envelope glycoprotein (Env) means for stabilizing Env trimer State- 1; and b. a pharmaceutically acceptable carrier.

14. A vaccine, comprising: a. a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer State- 1; and b. a pharmaceutically acceptable carrier.

15. A vaccine, comprising: a. a nucleic acid sequence encoding a modified human immunodeficiency virus- 1 (HIV-1) envelope glycoprotein (Env) trimer means for stabilizing Env trimer State- 1; and b. a pharmaceutically acceptable carrier.

16. The immunogenic composition of any one of claims 1-2, 4-6, 10, or 12-13, formulated as a mammalian vaccine.

17. The immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, or 16, formulated as a human vaccine.

18. The immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, or 16-17, wherein the Env variant trimer has a half life of 16 days or more at 0°C.

19. The vaccine of any one of claims 3, 7-9, 11, or 14-15, wherein the modified HIV-1 Env trimer has a half life of 16 days or more at 0°C20. The immunogenic composition any one of claims 1-2, 4-6, 10, 12-13, or 16-18, wherein the modified HIV-1 Env trimer contacted with a small molecule CD4 mimetic has: a. an IC50 of 150 pM or more; and b. a half-life of 9 days or more at 0°C.

21. The immunogenic composition of any one of claims 3, 7-9, 11, 14-15, or 19, wherein the modified HIV-1 Env trimer contacted with a small molecule CD4 mimetic has: a. an IC50 of 150 pM or more; and b. a half-life of 9 days or more at 0°C.

22. The immunogenic composition of claim 1, wherein the modified HIV-1 Env trimer is more sensitive to broadly neutralizing antibodies than the parental Env.

23. The vaccine of claim 1, wherein the modified HIV-1 Env trimer is more sensitive to broadly neutralizing antibodies than the parental Env.

24. A vaccine comprising an immunogenic effective amount of: a. the immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, 16-18, 20, or 22; or the vaccine of any one of claims 3, 7-9, 11, 14-15, 19, or 23; and b. an adjuvant.

25. A method of treating human immunodeficiency virus-1 (HIV-1), comprising administering: a. the immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, 16-18, 20, or 22; or b. the vaccine of any one of claims 3, 7-9, 11, 14-15, 19, or 23-24 in a subject with or at risk of having HIV-1.

26. Use of (a) the immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, 16-18, 20, or 22; or (b) the vaccine of any one of claims 3, 7-9, 11, 14-15, 19, or 23-24, for treating human immunodeficiency virus-1 (HIV-1).

27. The (a) immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, 16-18, 20, or 22; or (b) vaccine of any one of claims 3, 7-9, 11, 14-15, 19, or 23-24, for use in therapy.

28. The (a) immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, 16-18, 20, or 22; or (b) vaccine of any one of claims 3, 7-9, 11, 14-15, 19, or 23-24, for use in treating human immunodeficiency virus- 1 (HIV-1).

29. A method of invoking an immune response, comprising administering: a. the immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, 16-18, 20, or 22; or b. the vaccine of any one of claims 3, 7-9, 11, 14-15, 19, or 23-24 in a subject with or at risk of having HIV-1.

30. A method of generating antibodies in a subject, comprising administering: a. the immunogenic composition of any one of claims 1-2, 4-6, 10, 12-13, 16-18, 20, or 22; or b. the vaccine of any one of claims 3, 7-9, 11, 14-15, 19, or 23-24 in a subject with or at risk of having HIV-1.

31. The method of claim 29, wherein the antibodies are broadly neutralizing antibodies.

32. The method of any one of claims 25 or 28-29, wherein the subject is a human subject.

33. The use of claim 26, wherein the subject is a human subject.

34. The immunogenic composition of claim 27, wherein the subject is a human subject.

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