WO2024173897A1 - Modeling and stabilizing human immunodeficiency virus (hiv) envelopes - Google Patents

Abstract

Aspects of the invention are drawn to compositions and methods for stabilizing Human Immunodeficiency Virus (HIV) envelopes.

Description

MODELING AND STABILIZING

HUMAN IMMUNODEFICIENCY VIRUS (HIV) ENVELOPES

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/446,875, filed on February⁷ 19, 2023, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

GOVERNMENT INTERESTS

[0004] This invention was made with government support under grant No. UM1 AI44371 and awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0005] Aspects of the invention are drawn to compositions and methods for stabilizing Human Immunodeficiency Virus (HIV) envelopes.

SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [ ], is named [ ] and is [ ] bytes in size.

BACKGROUND OF THE INVENTION

[0007] A series of structural transitions in HIV-1 Envelope glycoprotein (Env) mediate host cell fusion. The Env is a trimer of heterodimers composed of gp!20 and gp41 domains responsible for receptor binding and membrane fusion, respectively. The host cell receptor CD4 induces rearrangements in gp!20 that expose the gp41 fusion machinery', leading from a closed to an open state (i.e. , conformational change). These transitions can occur independently of receptor engagement.

[0008] HIV-1 is a lentivirus that makes use of a class I viral fusion protein, called Env, to gain entry' into host cells to begin the process of integration and replication (Allan JS, et al. 1985. Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:1091-4). Like other class I viral fusion proteins, Env is proteolytically processed into tyvo distinct subunits, gpl20 and gp41 (Willey RL, et al. 1988. Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gpl60. Proc Natl Acad Sci U S A 85:9580-4). These subunits remain associated and oligomerize with other protomers to form the functional prefusion conformation of Env. which is composed of a trimer of gpl20/gp41 heterodimers. The N-terminal gpl20 subunit is responsible for mediating binding to both CD4 and CCR5 (Dalgleish AG, et al., 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312:763-7; Klatzmann D, et al. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 312:767-8; and Alkhatib G. et al.. 1996. CC CKR5: a RANTES, MIP-1 alpha, MIP-lbeta receptor as a fusion cofactor for macrophage-tropic HIV- 1. Science 272:1955-8), yvhile the gp41 subunit contains the hydrophobic fusion peptide and the helical heptad repeats that drive membrane fusion (Kowalski M, et al., 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237: 1351-5).

[0009] Due to the role that Env plays during the process of infection, it is the main target for vaccine development. However, several characteristics of Env make it a complex and problematic immunogen. To begin, it is covered by a dense glycan shield composed of N- linked glycans that hamper the elicitation of neutralizing antibodies (Wei X, et al. 2003. Antibody neutralization and escape by HIV-1. Nature 422:307-12.). Furthermore, constant immune pressure (Roark RS, et al. 2021. Recapitulation of HIV-1 Env-antibody coevolution in macaques leading to neutralization breadth. Science 371) and the infidelity of the HIV-1 reverse transcriptase has resulted in sequence diversity among Env variants (Patel PH & Preston BD. 1994. Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. Proc Natl Acad Sci U S A 91 :549-53). Env has also evolved to be metastable in the prefusion conformation, such that it is primed to rapidly and irreversibly transition to the postfusion conformation (Weissenhom W, et al., 1996. The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gpl20 and the N-terminal fusion peptide. EMBO J 15:1507-14.). This inherent metastability makes it difficult to recombinantly express the Env ectodomain in the antigenically desirable prefusion conformation.

[0010] In an effort to stabilize the prefusion conformation of the Env ectodomain without altering its antigenicity, Sanders et al. developed the SOSIP mutations, composed of an engineered disulfide bond (SOS) linking the gpl20 and gp41 subunits and an I559P (IP) substitution that disfavors formation of elongated postfusion helices that make up the six- helix bundle (Sanders RW, et al., 2002. Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J Virol 76:8875-89). Since their initial description, the SOSIP mutations have undergone iterative improvements to enhance the thermostability and the antigenic characteristics of the prefusion trimer (de Taeye SW, et al. 2015. Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes. Cell 163: 1702-15; Del Moral- Sanchez I, et al. 2022. High thermostability improves neutralizing antibody responses induced by native-like HIV-1 envelope trimers. NPJ Vaccines 7:27; and Torrents de la Pena A, et al. 2017. Improving the Immunogenicity of Native-like HIV-1 Envelope Trimers by Hyperstabilization. Cell Rep 20: 1805-1817). Alternative and complimentary protein engineering approaches have also been described as a means of yielding improved Env immunogens (Henderson R, et al. 2020. Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements. Nat Commun 11 :520; Kong L, et al. 2016. Uncleaved prefusion-optimized gpl40 trimers derived from analysis of HIV-1 envelope metastability. Nat Commun 7: 12040; Kwon YD, et al. 2015. Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env. Nat Struct Mol Biol 22:522-31; Rutten L. et al. 2018. A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers. Cell Rep 23:584-595; and Leaman DP & Zwick MB. 2013. Increased functional stability' and homogeneity of viral envelope spikes through directed evolution. PLoS Pathog 9:el003184). However, despite these advancements in immunogen engineering, many Env variants remain recalcitrant to recombinant in vitro expression.

SUMMARY OF THE INVENTION

[0011] HIV Env can exist in a prefusion (i.e., closed) conformation and can spontaneously and irreversibly transition to a postfusion conformation. In some embodiments, it is desirable that an Env vaccine antigen be stabilized in a prefusion conformation. While structures for prefusion and postfusion Env are available, steps in the transition from prefusion to postfusion conformation have not been determined. Herein, biophysical techniques (e.g., time resolved, temperature-jump (TR, T-Jump) small angle X-ray scattering (SAXS)) wasused to study this transition, to model structural intermediates in the transition process, and to use the structural intermediates to identify and make mutations in Env to stabilize it in a prefusion/ closed conformation.

[0012] Examination of a SOSIP stabilized Env gp!40 ectodomain revealed the rapid formation of a structural intermediate that moves to a distinct structural intermediate after ~750ps and rapid, laser induced system heating. Structure based modeling of these transitions indicated the structural states correspond to a single gpl20 protomer movement followed by movement of additional gpl20 protomers. Using these data, an inter-protomer apex stabilized construct was designed to eliminate access to these intermediates and therefore antibodies that target them. The data indicate that Env conformational transitions are complex and involve short-lived intermediate states that can be targeted for vaccine design.

[0013] Disclosed herein are modified HIV Env proteins that are modified so that, when the proteins are part of an Env trimer. the capability of the trimer in a closed or prefusion state, to form one of the structural intermediate states described above, is decreased. In some embodiments, modifications to HIV envelope proteins to accomplish this can stabilize V1/V2 interprotomer contacts at an apex of an Env trimer. In some embodiments, the modifications can be mutations in Env proteins. In some embodiments, the mutations link protomers in an Env trimer.

[0014] Disclosed herein are Env trimers that have 1, 2 or 3 of the modified Env proteins described herein.

[0015] Disclosed are vaccine compositions containing modified Env proteins or Env trimers containing one or more of the modified Env proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Certain illustrations, charts, or flow charts are provided to allow for a better understanding for the present invention. It is to be noted, however, that the drawings illustrate only selected embodiments of the inventions and are therefore not to be considered limiting of scope. Additional and equally effective embodiments and applications of the present invention exist.

[0017] FIG. 1 shows cartoon diagrams of HIV- 1 Env trimer transitioning from a closed to open state. gpI20, gp41, V1/V2, V3, and b20-b21 are colored blue, orange, green, red, and yellow, respectively. One subunit is omitted for clarity. These allosteric elements shown here rearrange during Env opening.

[0018] FIGS. 2A-C (A) shows a graph of non-limiting, exemplary binding data of antibody 17b to CH505TF SOSIP Env at 25°C (blue) and 50°C (red). Env has higher 17b binding at 50°C. (B) shows SAXS scattering difference curves for CH505TF Env at 25°C and 50°C. (C) shows theoretical SAXS difference scattering curves calculated from atomic models of a closed Env structure and an open Env structure.

[0019] FIGS. 3A-B (A) shows TR, T-Jump SAXS Curves for CH505TF Env at 1.5ms (red), 3ms (red-orange), 5ms (duplicates, orange and yellow-orange), lOus (y ellow), 50us (light green), lOOus (green), 300ms (teal), 500ms (sky blue), 1ms (triplicates, light blue, blue, dark blue), 10ms (purple), and 100ms (violet). Standard errors of the mean are shown in shaded colored regions. (B) shows the area under the scattering curves for each time point transition kinetics were determined from a double exponential decay model (black line).

[0020] FIGS. 4A-B (A) shows theoretical SAXS scattering difference curves for CH505TF Env. Difference curve for 3 protomers closed and 3 protomers open is shown in blue, and the difference curve for 1 protomer open and 3 protomers closed is shown in red. (B) shows a schematic proposed transition pathway for Env opening depicted in the same cartoon style as in FIG. 1. The 1 open occluded structure is a possible intermediate during Env opening.

[0021] FIG. 5 show s a ribbon diagram of the top-down view of the trimer apex.

[0022] FIG. 6 show s a non-limiting, exemplary SDS-PAGE gel of non-reduced and reduced samples.

[0023] FIG. 7 show s a non-limiting, exemplary SEC chromatogram.

[0024] FIG. 8 show s nano differential scanning fluorimetry (nanoDSF) thermogram.

[0025] FIGS. 9A-D shows negative stain electron microscopy (NSEM) micrographs of the sample image (A), sample image with a computer-assisted "Autopick" algorithm applied (B), 2D classification of autopicked particles (C), and an estimation of trimerized particles (Panel D)

[0026] FIGS. 10A-B shows HIV-1 envelope glycoprotein is structurally dynamic. (A) Linear sequence of HIV-1 Env with gp!20 in blue and gp41 in light orange and the layer-1, layer-2, variable domains 1 and 2 (V 1/V2), variable domain 3 (V3), (320- 21, fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), membrane proximal external region (MPER), transmembrane domain (TM) and the cytoplasmic tail (CT) allosteric elements are colored in dark blue, purple, green, red, yellow, sky blue, orange, dark orange, brown, tan. and grey, respectively. Glycosylation sites are denoted by grey forks. The location of SOS and IP mutations are also indicated. The black dashed line represents the location of truncation in SOSIP constructs. (B) Cryo-EM structures of a closed Env trimer (left) and an occluded Env trimer (middle,) fand and open Env trimer (right) from the side viewpoint. The boxes define the insets shown below. gp!20, gp41 and the allosteric elements are colored identically to panel A.

[0027] FIGS. 11A-D shows static small angle X-Ray scattering profiles capture HIV-1 Env opening. (A) Biolayer Interferometry (BLI) binding studies of 17B binding to BG505 Env SOSIP (blue) and CH505 transmitted founder (CH505TF) Env SOSIP (red). (B) Static small angle X-ray scattering (SAXS) difference curve for CH505TF and BG505 Env SOSIPS. (C) BLI binding studies of 17B binding to CH505TF Env SOSIP at 25°C (blue) and 50°C (red). (D) Static SAXS difference curves for CH505TF Env SOSIP at 50°C and 25°C.

[0028] FIGS. 12A-G shows time resolved, temperature-jump SAXS of HIV-1 Env Reveals two opening ransitions. (A) TR, T-Jump SAXS scattering difference curves for 1.5ps (red) 3ps (orange), 5ps (light orange lOps (yellow), 50ps (green), lOOps (cyan), 500ps (blue), 1ms (indigo), 10ms (violet), and 100ms (magenta) time delays. Dashed grey line at q=0.07A^-1 indicates the location of the feature of interest. (B) Singular value decomposition left vectors

1 (LV1, blue) and 2 (LV2, red). Dashed grey line at q=0.07A^-1 indicates the location of the feature of interest. (C) Deconvolved TR, T-Jump SAXS components 1 (blue) and component

2 (red) from REGALS decomposition SAXS difference curves in panel B. Dashed grey line at q=0.07A^-1 indicates the location of the feature of interest. (D) REGALS pair distance distribution for component 1 (blue) and component 2 (red). (E) The SVD right vectors 1 (RV1, blue) and 2 (RV2, red) showing the contribution of LV1 and LV2 at each time delay. The point show n as a red star was left out of the SVD fit to RV2. (F) The predicted concentrations of REGALS component 1 (blue) and component 2 (red). (G) The area under the curve (AUC) calculated according to Simpsons Rule for the TR, T-Jump SAXS difference curves shown in panel A and fit to a double exponential decay function.

[0029] FIGS. 13A-E shows theoretical SAXS profiles of glycosylated Env models reveal a one protomer open intermediate during Env opening. (A) Theoretical difference curves for 3Occluded-3Closed (red), 3 Occluded- 1 Occluded (blue), and lOccluded-3Closed (yellow). (B) The theoretical pair distance distribution for 3Closed (blue), 1 Occluded (yellow), 3Occluded (blue), and 3Open (green) Env conformations. (C) Theoretical difference curve for 3 Open-3 Occluded Env conformations. (D) The SVD first (blue) and second (red) left vectors for Man9 glycosylated modes (solid lines) and non-glycosylated models (dashed lines). (E) The distribution of RMSD over 250 5- .s classical MD simulations at 50°C for the gpl20 core b-sheets (blue), the V 1/V2 domain (green), and the V3 domain (red).

[0030] FIGS. 14A-E shows interprotomer disulfide bonds stabilize the closed Env trimer. (A) Cryo-EM density map of CH505.M5 Env SOSIP containing C127-C167 disulfides to lock the V1/V2 trimer contacts. (B) (C) (D) (E).

[0031] FIGS. 15A-E shows different HIV-1 Env SOSIP isolates have differences in SAXS profiles. (A) Static SAXS profiles for BG505 Env SOSIP (blue) and CH505TF Env SOSIP (red). The scattering intensity is expressed on the y-axis as a function of the scattering vector in A'¹. The box shows the region of the scattering profile displayed in the inset. (B) The Kratky plots for BG505 SOSIP (blue) and CH505TF SOSIP (red). (C) Guinier analysis of CH505TF Env SOSIP SAXS profiles. (D) The Guinier analysis of BG505 Env SOSIP SAXS profiles. (E) The pair distance distribution (P(r)) for BG505 Env SOSIP (blue) and CH505TF Env SOSIP (red).

[0032] FIGS. 16A-G shows HIV-1 Env SOSIP is stable under SAXS at elevated temperatures. (A) Static SAXS profiles for CH505TF Env SOSIP at 25°C (blue). 35°C (green), 44°C (yellow), and 50°C (red). The scattering intensity is expressed on the y-axis as a function of the scattering vector in A'¹. The box shows the region of the scattering profile displayed in the inset. (B) The Kratky⁷ CH505TF SOSIP at 25°C (blue), 35°C (green), 44°C (yellow), and 50°C (red). (C) Guinier analysis of CH505TF Env SOSIP SAXS profiles at 25°C. (D) The Guinier analysis of CH505TF Env SOSIP SAXS profiles at 35°C. (E) The Guinier analysis of CH505TF Env SOSIP SAXS profiles at 44°C. (F) The Guinier analysis of CH505TF Env SOSIP SAXS profiles at 50°C. (G) The pair distance distribution (P(r)) for CH505TF Env SOSIP at 25°C, 35°C, 44°C, and 50°C in blue, green, yellow, and red, respectively.

[0033] FIGS.17A-E shows CH505TF Env SOSIP has time-dependent changes in SAXS profiles. (A) Experimental protocol for the pump-probe set up. The horizontal black arrow represents the progression of time during the experiment. The grey bars represent the X-ray probe step and the red bars represent the infrared laser pump step. For the time resolved (TR) temperature-jump (T-jump) SAXS experiments, the X-ray scattering was collected at 2 time points (-10ms and -5ms) prior to the infrared laser pump and then the IR pump step is interleaved with the X-ray probe at various time delays after the IR pump. The time delays measured for this experiment range from 1.5ms to 100ms as well as 500ns (B) TR, T-Jump SAXS scattering difference curves for 1.5ps (red) 3ms (orange), 5ps (light orange I Ops (yellow), 50ps (green), lOOps (cyan), 500ps (blue), 1ms (indigo), 10ms (violet), and 100ms (magenta) time delays. Dashed grey line at q=0.07 A" ¹ indicates the location of the feature of interest. (C) The Kratky plots for CH505TF Env SOSIP at 1.5ps (red) 3ps (orange), 5ps (light orange lOps (yellow), 50ps (green), lOOps (cyan), 500ps (blue), 1ms (indigo), 10ms (violet), and 100ms (magenta) time delays. (E) The Guinier analysis of CH505TF Env SOSIP SAXS profiles at 44°C. (F) TR, T-Jump SAXS scattering difference curves for 500ns.

[0034] FIGS.18-A-B shows calibration of TR, T-Jump. (A) Static SAXS scattering difference curves for A5 (blue), A6 (yellow), A 8 (red), and lOps H2O T-Jump (grey). The change in scattering intensity is plotted as function of the scattering vector in A’¹. (B) Linear fit of the difference curves in panel A. The maximum water scattering intensity is plotted as a function of the temperature difference. The grey circles represent the experimental data, and the red line is the linear regression fit to the experimental data. Estimates for the lOps H2O T- Jump, 1.5ps CH505TF Env SOSIP T-Jump, 10ms CH505TF Env SOSIP T-Jump, and the 100ms CH505TF Env SOSIP T-Jump are shown by the green star, yellow square, blue triangle, and purple diamond, respectively.

[0035] FIGS. 19A-D shows decomposition of CH505TF Env SOSIP TR, T-Jump SAXS difference profiles. (A) The singular values of SVD on CH505TF Env SOSIP TR, T-Jump difference profiles plotted as a function of the extracted SVD vector. (B) CH505TF Env SOSIP TR, T-Jump SVD left vectors 3-8. (C) CH505TF Env SOSIP TR, T-Jump SVD right vectors. (D) ² values of the REGALS fit to the TR, T-Jump SAXS data.

[0036] FIGS. 20A-E shows CH505TF Env SOSIP Model Glycosylated with Mannose-9. All-atom model of CH505TF Env SOSIP glycosylated with Man9 in the closed (A), lOccluded (B), 3Occluded (C), and 3Open conformations (D). (E) The theoretical scattering profiles showing the scattering intensity in the log scale as a function of the scattering vector in A’¹ for Man9 glycosylated 3Closed (blue), lOccluded (yellow), 3Occluded (red), and 3Open (green) CH505TF Env SOSIP models. The box shows the region detailed in the inset. [0037] FIGS. 21A-D show (A) Differential fluorescence temperature induced melting profiles for the parent design as well as pre and post V3-antibody negative selection-based purification. (B) SDS-PAGE gel of (1) protein molecular weight marker (2) Parent, (3) pre V3-negative selection design, (4-6) sequential iterations of V3-negative selection, (7 and 8) V3-negative selection eluate, (9-15) reduced samples of (3-8). (C) Biolayer interferometry binding responses for 17B, 19B, and PGT151 for the parent and the design in the presence and absence of CD4-Ig. (D) Negative stain electron microscopy-based 3D reconstruction of the design. [0038] FIGS. 22A-F show (A) (top) Example cryo-electron micrograph highlighting selected design particles, (bottom) 2D classes of the design trimer. (B) (top) Open state 3D map of particles isolated by 3D-classificaiton of the design dataset, (bottom) FSC plot for the open state reconstruction. (C) Closed state design structure aligned with the open state map. (D) FSC plot for the closed state reconstruction. (E) Example coordinate fit to the design closed state map. (F) Local resolution map of the design closed state.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Disclosed here are compositions and methods for stabilizing the HIV-1 Env trimer. Aspects of the invention are drawn towards mutations and modifications for stabilizing HIV- 1 Env trimers, models for determining mutations for stabilizing HIV-1 Env trimer. models for determining vaccine targets, vaccines for HIV-1, anti-bodies against HIV-1, and methods of using the same.

[0040] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0041] The singular forms "a", “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [0042] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0043] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0044] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a. b and c. Wherever the terms “a” or '‘an’’ are used, ‘'one or more’’ is understood, unless such interpretation is nonsensical in context.

[0045] As used herein, the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. The term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

Env Prefusion Forms, Intermediate Forms, Postfusion Forms, and Protein Instability [0046] Disclosed herein are biophysical studies of HIV envelope proteins (Env) that led to a model for how the proteins transition from a closed, prefusion conformation to a postfusion conformation. The biophysical studies led to predicted, transient intermediate states (e.g., 1 open occluded and open) during the transition of the closed prefusion state to the postfusion state. From the intermediates, we have predicted and made mutations in Env that prevent it from forming the intermediates and transitioning to the postfusion state. Disclosed are mutations and modifications for stabilizing HIV-1 Env trimers, models for determining mutations for stabilizing HIV-1 Env trimers, models for determining vaccine targets, vaccines for HIV-1, antibodies against HIV-1, and methods of using the same.

[0047] The Env proteins disclosed herein can be from HIV. The HIV-1 Env can be from Group M and from subtype or clade A, B, C, D, F, G, H, J or K. In some embodiments, the mutations can be mutations to viral envelope proteins (Env). The Env protein can be an ectodomain. The Env protein can be a gpl60, a gpl20, or a gp41 protein. The Env proteins can comprise V1/V2, V3, and 1320-1321.

Modeling and Modifications to Env

[0048] Described herein are models for determining mutations or modifications to stabilize the HIV-1 Env trimer. In some embodiments, the model can comprise intermediate states of the HIV-1 Env trimer. As used herein, the term “intermediate” state can refer to a transitional state. As used herein, the term “state” and “configuration” can be used interchangeably. [0049] As used herein, the term “intermediate” can refer to a state between two states. In some embodiments, the intermediate can refer to a state between an initial state and a subsequent state. In embodiments, the initial state is the prefusion state. In embodiments, the subsequent state is the postfusion state. In embodiments, the prefusion state can comprise the "closed" state.

[0050] In embodiments, the intermediate state can be an intermediate energetic state. For example, the intermediate state can be a metastable state. In embodiments, the intermediate can be a transition state. As used herein, the term ‘‘transition state” can refer to the configuration at a local energy maximum.

[0051] Aspects of the invention are drawn towards a model for determining mutations or modifications to the HIV-1 Env trimer to prevent changes in the V1/V2 region and rotation of the gpl20 domains from taking place. Without wishing to be bound by any particular theory, these models can identify the designs that lock the trimer into the closed position. For example, a design that locks the trimer closed can link the trimer apex across protomers to fully lock the trimer closed. In embodiments, vaccine targets can be identified from these models.

[0052] In some embodiments, the mutations can be made in VI and/or V2 loop regions of Env. In some embodiments, the mutations can be made in V3 loop regions of Env. In some embodiments, the mutations can be made in the P20-J321 regions of Env. In some embodiments, the mutations (e.g., disulfide bonds) can connect or link individual monomers or protomers in the trimeric Env structure. In some embodiments, the mutations can connect or link protomers across the apical region of a trimeric Env structure (FIG. 5).

[0053] Aspects of the invention are drawn towards mutations and modifications. In some embodiments, the mutations disclosed herein have substitutions of certain amino acids that are found in the naturally occurring or wild-type forms of the proteins. In some embodiments, the amino acid positions that are substituted can be positions in a protein that are involved in a prefusion conformation of the protein changing to a postfusion conformation. Generally, modification of the protein by substitution of amino acids at these locations disfavors conformational change of the modified protein from a prefusion to postfusion state as compared to the unmodified protein (e.g., disfavors a-helices).

[0054] In embodiments, the mutations can result in interactions, bonds, or a combination thereof which can stabilize the trimer apex. For example, the interactions, bonds, or combinations thereof can comprise disulfide bonds, salt bridges, glycine linkers, or a combination thereof.

[0055] As used herein, the term “disulfide bond” can refer to a bond formed from the coupling of two thiol groups. In embodiments, the disulfide bond can be the product of a mutation or modification which results in the formation of cystine. For example, the mutation can comprise substituting a non-cysteine amino acid for cysteine. For example, the modification can comprise the oxidation of two thiol end groups.

[0056] As used herein, the term “salt bridge” can refer to an interaction between two groups of opposite charge in which at least one pair of heavy atoms is within hydrogen bonding distance. In embodiments, the salt bridge can comprise an interaction between an anionic group and a cationic group. For example, the anionic group can comprise the carboxylate of aspartate or glutamate. For example, the cationic group can comprise a cationic ammonium of lysine or a guanidinium of arginine. In some embodiments, positively charged histidine, tyrosine, and serine groups can participate in a salt bridge.

[0057] These mutations and modifications can generate modified proteins that can have properties that are different than the unmodified protein from which the modified proteins are derived. In various embodiments, the modified proteins can be more likely to retain a prefusion conformation and less likely to assume a postfusion conformation as compared to the unmodified protein from which the modified protein is derived. In embodiments, the modified protein can be more stable than the unmodified protein. In embodiments, the modified protein can be produced in higher yields than the unmodified protein under similar experimental conditions. In some embodiments, increased stability can result in improved protein yield in recombinant expression systems. In some embodiments, the modified proteins can have longer half-life in the body of an individual who has been administered the protein (e.g.. as a vaccine).

Vaccine Compositions

[0058] Herein, vaccine compositions refer to compositions of the modified proteins described herein suitable for administration to an individual for the purpose of prophylactically or therapeutically protecting the individual against, for example, an HIV-1, HIV-2 or SIV infection. Generally, the vaccine compositions contain prophylactically- or therapeutically-effective amounts of the modified proteins. In some embodiments, the vaccine compositions can include pharmaceutically acceptable carriers, diluents or excipients. The vaccine compositions can be administered to an individual by various routes, including oral, subcutaneous, parenteral (such as, intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocular and pulmonary administration.

Methods [0059] Disclosed are methods for generating models of HIV- 1 to determine mutations and modifications to stabilize the HIV - 1 Env trimer. In embodiments, the method comprises identifying an intermediate state of the Env trimer. In embodiments, identifying an intermediate state of the Env trimer can comprise performing biolayer interferometry to correlate differences in small angle scattering (SAXS) profiles of Env conformations, performing time-resolved, temperature-jump small-angle X-ray scattering (TR. T-jump SAXS). After the intermediate state is identified, the mechanistic details regarding conformational changes from the prefusion state to the postfusion state can be elucidated to generate a model for determining mutations and modifications to stabilize the Env trimer. In some embodiments of the methods, amino acids within the proteins are selected for amino acid substitution based on the position and/or likely function of the amino acid in the protein. For example, amino acids that can play a role in the conformational change can be selected. In some embodiments, amino acid positions can be selected that, if substituted with a different amino acid, can decrease the probability that a prefusion conformation of the Env protein will change to a 1 open occluded, open and/or postfusion conformation.

[0060] In some embodiments, the substituting amino acid (i.e. , the amino acid that replaces the amino acid in the unmodified protein) can prevent or decrease the probability that the protein, when in the prefusion conformation will transition to a 1 open occluded, open and/or postfusion conformation.

Methods Used in Examples

[0061] Sample Preparation

[0062] CH505 transmitted founder (CH505TF) HIV-1 Env SOSIP was produced according to previously published methods. CH505TF protein was concentrated to a final concentration of 5. 125mg/mL in 15mM HEPES buffer with 150mM NaCl, for a total volume of ~4 mL. Protein sample was syringe filtered with a 0.22-micron filter and degassed before each use. At the end of each day, protein sample was removed from the flow cell and stored at 4°C overnight.

[0063] TR, T-Jump Beamline Setup

[0064] The BioCARS 14-ID-B beamline at the Advanced Photon Source at Argonne National Laboratory was used to conduct time resolved, temperature-jump small angle X-ray scattering (TR, T-Jump SAXS) and static temperature series SAXS experiments. A peristaltic pump injected protein/buffer solution into a 700-micron quartz capillary mounted on a custom, temperature-controlled aluminum nitride (AIN) holder. Temperature jumps of the sample were achieved using a 7ns infrared (IR) laser at 1.443 micrometers and l.lmJ/pulse to excite the O-H stretch in water, resulting in an approximately 6°C temperature jump (FIG. 18B). After a variable time delay, sample was probed with a pink X-ray beam with photon energy 12keV (FIG. 17A). Images were collected using a Rayonix MX340-HS x-ray detector in scattering vector (q) range 0-2.5A'¹.

[0065] Temperature Jump Calibration

[0066] We measured the static SAXS signal of water every 3°C from 25°C to 51°C. We calculated static SAXS difference curves for A5°C, A8°C, and Al 1°C by subtracting the SAXS signals at 45°C and 40°C, 48°C and 40°C, and 51°C and 40°C, respectively (FIG. 18A). We then measured the TR, T-Jump SAXS signal of water at lOps after heating, starting at 42°C. The temperature jump was determined to be A6°C by linear regression analysis of the water TR, T-Jump and static SAXS difference signals (FIG. 18B).

[0067] Static SAXS

[0068] We measured the static SAXS scattering profiles of BG505 and CH505TF Env SOSIP at 25°C in the scattering vector range q=0-0.8A^-1 (FIG. 15A).

[0069] Static SAXS Temperature Series

[0070] We measured the static SAXS of both 15mM HEPES buffer and of 5.125mg/mL CH505TF sample at 25°C. 35°C, 44°C, and 50°C (FIG. 16A). Static SAXS signals were collected using 24 bunches in continuous translation mode at 20Hz on the BioCARS 14-ID-B beamline. 50 images were collected for each set for scattering vector range q=0-2.5A'¹.

[0071] CH505TF Time Delay Series

[0072] We measured the TR, T-Jump SAXS signal for 5.125mg/mL CH505TF SOSIP sample at several different time delays after sample heating from 44°C to 50°C by IR laser: 1.5ps, 3. Ops, 5. Ops, lOps, 50ps, lOOps, 500ps, 1ms, 10ms, and 100ms. For each time delay measured, TR, T-Jump SAXS was also measured at -lOps and -5ps prior to heating with IR laser (‘laser off; FIG. 17A). To accommodate the large change in timescales and minimize systematic errors due to experimental drift, we measured TR, T-Jump SAXS for buffer and Env in sets, with overlapping time delays between each set. The CH505TF Env SOSIP TR, T-Jump SAXS was measured in three sets. Env set 1 includes -lOps, -5ps, lOps, 50ps, lOOps, 500ps, and 1ms measured with 24 bunches in continuous translation mode at 20Hz, collecting 250 images for each time delay. Set 2 for CH505TF Env SOSIP was measured with 11 bunches in 20Hz continuous translation mode and includes -lOps, -5ps, 1.5ps. 3ps, and 5ps, with 362 images for each time delay. 200 images were collected for Env set 3, which was measured in 5Hz step translation mode with 24 bunches and included time delays -lOps, -5ps, 1ms, 10ms, and 100ms.

[0073] TR, T-Jump SAXS profiles of HEPES were collected using the same protocol. For HEPES set 1, 100 images each at -lOps, -5ps, 1.5ps, 3. Ops, and 5. Ops time delays were collected with 11 bunches in 20Hz continuous translation mode. HEPES set 2 was collected with 24 bunches in 20Hz continuous translation mode and consists of 50 images each at time delays -5ps, -lOps. 5ps, lOps. 25ps. 50ps, lOOps. 250ps. 500ps, and 1ms. HEPES set 3 was measured in 25 bunches in 5Hz step mode, collecting 50 images each at -lOps, -5ps, 1ms, 10ms, and 100ms time delays.

[0074] Data Reduction

[0075] Scattering intensity (I) was binned as a function of scattering vector (q, A ¹) and radially averaged to produce isotropic scattering curves I(q) vs. q. q is calculated according to Eq. 1.

[0076] Q = 47T ^ (Eq. 1)

[0077] where 2q is the scattering angle and 1 is the X-ray w avelength. A mask w as applied below 0.02A'¹ q to eliminate scattering signal due to beam stop for all collected images for static and TR, T-Jump SAXS data sets collected on the BioCARS 14-ID-B beamline. The scattering curves w ere normalized to the isobestic point for w ater. This data reduction was performed with custom softw are at the BioCARS beamline.

[0078] Data Processing

[0079] For static SAXS scattering curves, outliers were detected using two iterations of singular value decomposition (SVD) to remove curves more than 2.5 standard deviations aw ay from the mean. Once outliers were removed, average curves for each temperature were calculated for both HEPES and CH505TF Env SOSIP samples. The procedure was identical for both HEPES and CH505TF Env SOSIP. The remaining curves were used to determine the average static curves for HEPES and CH505TF Env SOSIP. The average HEPES buffer curves were scaled to the average CH505TF curves in the scattering vector range q=1.5-2.5A" \ with the scaling factors (s) calculated according to Eq. 2.

[0081] The scaled average HEPES buffer scattering curves w ere subtracted from the average CH505TF static scattering curves for each temperature to produce buffer-corrected average static SAXS scattering curves. [0082] To determine outliers for TR, T-Jump SAXS curves, two iterations of SVD on both laser on and laser off scattering curves was used to detect scattering curves more than 2.5 times standard deviations from the mean. This outlier analysis was carried out for - 1 Ops and - 5ns laser off scattering profiles as well as the scattering profiles for each time delay after heating with the IR laser. Difference curves were then calculated for the remaining scattering curves by subtracting the - I Ops laser off curve from each time delay. An iterative / ² analysis was used to detect difference curves with a / ² > 1.5 for TR, T-Jump SAXS at each time delay. The same procedure was performed on both HEPES buffer and CH505TF Env SOSIP. Average difference curves for each time delay were calculated from the remaining difference curves. For each time delay difference profile, the corresponding average buffer difference curves were scaled using Eq. 2 to the average CH505TF Env difference curves in the buffer scattering region (q=1.5-2.5A‘¹) and subtracted from the respective average CH505TF difference profiles to yield average, buffer-corrected TR, T-Jump SAXS difference curves for CH505TF Env SOSIP. These buffer-subtracted curves were used for the remaining TR, T- Jump and static SAXS data analysis.

[0083] Average buffer-subtracted CH505TF scattering profiles were determined using the laser-on scattering curves for CH505TF Env SOSIP and HEPES buffer T-Jump scattering curves. The same procedure was followed for determining these buffer-subtracted T-Jump scattering curves as for the static SAXS curve processing described above.

[0084] All data processing was carried out in custom code implemented in Python3. TR, T- Jump Kinetic Analysis

[0085] The area under the average difference curve (AUC) for each CH505TF Env time delay was determined according to Simpsons rule. The q range 0.02-0. 1 A^-1 of the average difference curves was used for AUC determination. The AUC vs. time delay plots were fit to a double exponential w i th the form shown in Eq. 3

[0086] y = a • (1 - e~^bx) + c ■ (1 - e~^dx) + e (Eq. 3)

[0087] where 6=l/tfast and d=l/tsiow.

[0088] AUC and fits to double exponential decay models were calculated in with custom code implemented in Python3.

[0089] TR, T-Jump Component Analysis

[0090] The average, buffer-subtracted CH505TF TR, T-Jump difference curves were all input to SVD decomposition implemented to extract individual signals composing the TR, T-Jump SAXS difference profiles. The singular values were used to determine the total number of component signals. The SVD left vectors reveal the individual difference curves contributing the TR, T-Jump SAXS difference profiles while the right vectors show the contribution of the corresponding left vector at each time delay and were fit to the double exponential decay function in Eq.3 to extract kinetic parameters for the individual SVD components.

[0091] An alternative decomposition method, REGALS was used to deconvolve the TR, T- Jump SAXS signals. We built a REGALS model of the CH505TF Env SOSIP TR. T-Jump data based on two components. For both components, a smooth parameterization was used for concentration basis and the real-space parameterizations were selected for SAXS difference profile basis Component 1 and component 2 used a maximum dimension (dmax) of 154 and 173A. respectively. No other constraints were applied to the REGALS model.

SAXS QC Analysis

[0092] Guinier analysis was performed in ATSAS primus software to assess for scattering artifacts caused by radiation damage for both static (FIG. 15C-D; FIG. 16C-F) and TR, T- Jump SAXS scattering data. Scattering artifacts due to Env aggregation were assessed with Kratky plots and pair-distance distributions (P(r)) for both static (FIG. 15B, E; FIG. 16B, G) and TR, T-Jump scattering profiles were also calculated in Primus.

[0093] Modeling

[0094] Atomic models of CH505TF Env in a 3 protomers closed (3Closed), 1 protomer open occluded (1 Occluded), 3 protomers open occluded (3 Occluded), and 3 protomers open (3Open) conformations were built using Modeller 10.1. 6UM6 was the template for closed protomers. Two different one open and 3 open protomer models were made, resulting in a total of 5 different Env models. Open protomer models were based on 6CM3 template while open occluded protomers were modeled based on 7TFN template. 6UM3 was used as the template for open models. Clustal Omega alignments between CH505TF Env SOSIP sequence and template structures were done using DNASTAR MegAlign software. Disulfide patches were added to Modeller scripts to ensure formation of disulfide bonds.

[0095] Glycosite was used to predict the likely glycosylation sites in CH505TF Env. Glycosylator was used to glycosylate these glycan sites with mannose-9 (Man9) glycans. Three rounds of refinement were performed to remove clashes. All rounds of refinement used 10 iterations, a 0.01 mutation rate, and a population size of 30. The first two rounds of refinement used 10 generations, while the last refinement used 20 generations. The number of individues was decreased by two between each refinement. [0096] The theoretical SAXS profiles for each glycosylated model were calculated using the FoXS server. P(r) distributions were for these theoretical SAXS scattering profiles were determined in ATS AS primus software and compared to the experimental and REGALS SAXS profiles. Theoretical difference profiles were calculated by subtracting 3Closed theoretical scattering profiles from lOccluded, 3Occluded, or 3Open theoretical scattering profiles.

[0097] Molecular Dynamics

[0098] The CHARMM-GUI Glycan Modeller was used to glycosylate glycosite-predicted glycosylation sites with mannose-5 glycans and prepare gpl20 systems for simulation. 250 5- ps trajectories were run for closed gp!20 at and 50°C, respectively. The Amber20 software package with the CHARMM36 force field and TIP3P water model was used for simulations. An 150A octahedral box was used to allow for V 1/V2 and V3 dislocation during simulation. Both systems were neutralized with 150mM NaCl. 2000 steps of steepest decent energy minimization was performed with release of protein backbone constraints after 1000 steps. Minimization was followed by 20 ps of NVT equilibration with protein atoms with a lOkcal/mol restraint and a subsequent 5 ns NPT equilibration with no restraints. Constant temperature of 323.15K was maintained using a Langevin thermostat and constant pressure of 1 bar was maintained by a Berendsen barostat. Electrostatic interactions were calculated with the Particle Mesh Ewald method with a cutoff of 12A and switching distance of lOA. The SHAKE algorithm was used to constrain covalent bond lengths. Unrestrained production simulations were performed for 5ms in the NPT ensemble.

[0099] RMSD Calculations

[00100] Cpptraj of AmberTools21 was used to determine the RMSD time series of V1/V2, V3, and gpl20 core b-sheets backbone for each replicate of MD simulation. A custom python script was used to determine the average RMSD and frequency distributions for all replicates aggregated.

EXAMPLES

[00101] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results. EXAMPLE 1- Examining HIV-1 Envelope Conformational Transitions at High Temporal Resolution

[00102] While Env trimer closed and open state structures are available, a step-by-step mechanism of structural motions connecting these states remains undetermined. Here, we measured Env transitions on the microsecond timescale to shed light on this process.

[00103] Methods

[00104] Binding of the open state. gp !20 bridging sheet interactive, CD4-induced (CD4i) antibody 17b to Env SOSIPs via biolayer interferometry was used to correlate differences in small angle X-ray scattering (SAXS) profiles with Env conformation. Time-resolved, temperature-jump small-angle X-ray scattering (TR, T-jump SAXS) was used to measure microsecond timescale dynamics of the SOSIP Env. Structural transitions were induced by a rapid, nanosecond timescale temperature jump facilitated by exposure to an infrared laser heating source. The structural state of the protein was probed at several time-delays after laser heating via SAXS measurement. Scattering data were collected at 11 different time points after heating: 1.5ps. 3. Ops, 5. Ops. lOps, 50ps, lOOps, 300ps. 500ps. 1ms, 10ms, and 100ms. Scattering difference curves were calculated by subtracting the pre-heating scattering signal from the post-heating scattering curve for each time point. Transition rates were determined using singular value decomposition and fitting of an exponential decay model to the first and second SVD right vectors.

[00105] Non-Limiting. Exemplary Results

[00106] A distinct feature in the SAXS curves for the BG505 and CH505 transmited founder (CH505TF) Env SOSIPs correlated with differences in 17b antibody binding. The CH505TF Env displayed differential scatering intensity at elevated temperatures and was therefore selected for the TR, T-jump measurements. The TR, T-Jump SAXS data resolved two distinct structural transitions. The first transition occurs with a time constant of ~5ps, with the second, slower transition, occurring with a rate constant of ~700ps. This data is consistent with a model of Env opening in which a transient, intermediate state forms rapidly during the initial stages of Env conformational changes followed by changes correlated with greater exposure of coreceptor binding site.

[00107] Non-Limiting, Exemplary Conclusions

[00108] Our results indicate that Env opening involves a short-lived, transient intermediate state. The early structural rearrangements captured here provide important mechanistic details regarding Env conformational dynamics with impacts on vaccine immunogen design. [00109] FIG. 1 shows a diagram depicting the change from a prefusion closed state to an open state, highlighting the changes in the V1/V2 region that take place and rotation of the gpl20 domains. Blocking these movements is a vaccine design target to ensure antibody responses to the open state that are not useful are not induced. This model (and in FIG. 4B) were based on the data obtained in the studies described below.

[00110] FIG. 2 Panel A shows binding of 17b antibody to CH505TF SOSIP at 25°C (blue) and 50°C (red). Env has higher 17b binding at 50°C. (Panel B) SAXS scattering difference curves for CH505TF Env at 25°C and 50°C. (Panel C) Theoretical SAXS difference scattering curves calculated from atomic models of a closed Env structure and an open Env structure.

[00111] We can identify transient intermediate states that have not been observed, and without wishing to be bound by any theory, are involved in the closed to open transition. As such a state can provide a model to further stabilize the trimer. This is desirable as recent studies have shown that off target antibody responses to open states in which gpl20 has rotated but the V1/V2 region remains unchanged can occur when immunizing with SOSIP trimer immunogens.

[00112] To see these short-lived states, we used a rapid (-250 nanoseconds) temperature jump that induces the trimer to change conformation. With this perturbation we can then monitor the changes that take place on the microsecond timescale. We can first indicate that the trimer responds to temperature. The results shown herein can support that the CH505TF SOSIP conformation is indeed sensitive to temperature and consistent with increased openness at elevated temperature.

[00113] TR, T-Jump SAXS Curves for CH505TF Env at 1.5ms (red). 3ms (red-orange), 5ms (duplicates, orange and yellow-orange), lOus (yellow). 50us (light green), lOOus (green), 300ms (teal), 500ms (sky blue), 1ms (triplicates, light blue, blue, dark blue), 10ms (purple), and 100ms (violet). Standard errors of the mean are show n in shaded colored regions (FIG. 3 Panel A). (FIG. 3 Panel B) Area under the scattering curves for each time point transition kinetics were determined from a double exponential decay model (black line).

[00114] These results indicate that the trimer responds to temperature on the microsecond timescale displaying two distinct transition timescales, including a short-lived intermediate that appears in less than ten microseconds after the rapid temperature jump.

[00115] Theoretical SAXS scattering difference curves for CH505TF Env. Difference curve for 3 protomers closed and 3 protomers open is shown in blue, and the difference curve for 1 protomer open and 3 protomers closed is shown in red (FIG. 4 Panel A). (FIG. 4 Panel B) Non-limiting, exemplary transition pathway for Env opening depicted in the same cartoon style as in FIG. 1. Without wishing to be bound by any theory, the 1 open occluded structure can be an intermediate during Env opening.

[00116] These results indicate that theoretical difference curves between a closed to open and a close to single protomer gpl20 rotated open (1 open occluded) can give rise to the difference curves obtained via SAXS. Our designs are based on this observation. That is. without wishing to be bound by theory’, the designs that lock the trimer closed, link the trimer apex across protomers to fully lock the trimer closed.

[00117] FIG. 5 shows a top-dow n view of the trimer apex with each protomer highlighted in a different color. The magenta sphere indicates non-limiting, exemplary locations of mutations to stabilize hold the apex together, ensuring it is unable to access the intermediate or open states. Mutations can comprise disulfide bonds between protomers, salt-bridge formation, and glycine linker addition to stabilize the backbone fold.

[00118] FIG. 6 illustrates an SDS-PAGE gel of various Env proteins. Lane 1 shows molecular weight standards. Lane 2 shows a non-reduced, unmodified Env protein. Lane 3 shows a non-reduced, Env protein that was modified to contain disulfide bonds to link protomers in an Env trimer. These data show that the protein in Lane 3 migrates more slowly on the gel than the protein in Lane 2, indicating a change in structure/conformation of the protein in Lane 2. Lane 7 shows the protein from Lane 3 in a reduced state. The protein in Lane 7 migrates faster than the protein in Lane 2, indicating that reduction of the protein broke the disulfide bonds in the protein.

[00119] FIG. 7 shows a non-limiting, exemplary’ SEC chromatogram.

[00120] FIG. 8 shows a nano differential scanning fluorimetry (nanoDSF) thermogram. [00121] FIG. 9 To obtain the micrographs in FIG. 9, a frozen aliquot from -80 °C was thawed at room temperature in Al block for 5 min. The sample was then diluted 1 : 1 to 270 pg/ml with 5 g/dl Glycerol in HBS (20 mM HEPES, 150 rnM NaCl pH 7.4) buffer containing 8 mM glutaraldehyde. After 5 min incubation, glutaraldehyde was quenched by adding sufficient 1 M Tris stock, pH 7.4, to give 80 rnM final Tris concentration and incubated for 5 min. The quenched sample was applied to a glow-discharged carbon-coated EM grid for 10- 12 second, then blotted, and stained with 2 g/dL uranyl formate for 1 min, blotted and airdried. Grids were examined on a Philips EM420 electron microscope operating at 120 kV and nominal magnification of 49,000x, and 14 images were collected on a 76 Mpix CCD camera at 2.4 A/pixel. Images were analyzed by 2D class averages using standard protocols with Relion 3.0 (Zivanov et al. 2018. eLife. 7:e42166). EXAMPLE 2 - Conformational Transitions of Modified Env Protein 1

[00122] To study conformation transitions in Env proteins that are modified to decrease Env transitioning to 1 open occluded, open and/or postfusion states, the modified, nonreduced Env protein, containing disulfide bonds that link protomers in an Env trimer, is used in an experiment similar to that which produced the data shown in FIG. 3A (TR, T-Jump SAX Curves). From the data from this experiment, we expect to conclude that the modified protein does not produce, produces a lower amount of, or produces an intermediate that is different than the short-lived intermediate produced by the unmodified protein that appears in less than 10 seconds after the rapid temperature, as shown in FIG. 3B (e.g., the open state of the protein).

[00123] To study antigenic landscape of the non-reduced Env protein containing disulfide bonds to link protomers, a panel of antibodies that bind to epitopes throughout Env are tested for binding to the modified Env and to an unmodified Env. The data are expected to show that the overall antibody binding characteristics of the modified Env are similar to the unmodified Env.

EXAMPLE 3 - Conformational Transitions of Modified Env Protein 2

[00124] Small angle X-rav scattering captures closed-to-open transitions in HIV-1 Env glycoproteins

[00125] Time-resolved, temperature-jump SAXS experiments are referred to as pumpprobe experiments. In the pumping stage, the system is perturbed, in this case by rapid heating of the water surrounding the sample by an infrared laser. The probe stage occurs at a time delay relative to the pump stage and acts as a readout for the state of the system, in this case a SAXS profile that reports on the structural state of the protein (FIG. 17A). The successful application of this method therefore requires a construct capable of readily changing conformational state with the temperature jump and that the conformational change can be detected by SAXS measurement. We therefore sought first to identify a structurally labile Env isolate suitable for TR, T-Jump SAXS experiments. We used biolayer interferometry (BLI) to measure conformation specific antibody binding to two SOSIP stabilized Env gp!40 ectodomains, CH505 and BG505, both isolated from infected individuals (FIG. 11A). The antibodies included the trimer specific PGT145 Mab, the coreceptor binding site targeting 17B Mab, and the V3 exposed targeting 19B Mab.

Envelopes that access the open configuration more readily bind 17B and 19B while binding of PGT45 confirms the presence of closed trimer. The results indicate the CH505 SOSIP interacts with both 17B and 19B while BG505 does not. Both show binding to PGT145 indicating both display a closed trimeric state. The CH505 SOSIP is therefore more structurally labile and may more readily respond to perturbation.

[00126] Small-angle X-ray scattering data is collected for proteins in solution and is therefore isotropic due to molecular movement. The resulting scattering curves therefore report low resolution features. It is therefore important to identify specific curve features that correlate with differences in specific structural features of interest. Owing to the differences in antibody binding between CH505 and BG505, we reasoned that a differentiating feature would be found between the two in a scattering profile. We measured static SAXS profiles for CH505 and BG505 SOSIPs (FIG. 15A) and calculated difference profiles by subtracting the BG505 Env SOSIP scattering curve from the CH505 Env SOSIP scattering curve (FIG. 11B). The difference profiles show a noticeable feature around a scattering vector (q) value of 0.07 A'¹ in the SAXS difference profiles with a downward trend between 0.01 and 0.05 A'¹ (FIG. 11B). We next compared each with a scattering profile from a stabilized version of CH505. The CH505 vs. stabilized CH505 and stabilized CH505 vs. BG505 difference curves show similar features and directionality that correlated with antibody binding (FIG. 15A). Together these results show that SAXS can indeed report on Env structural differences related to the propensity to access states downstream from the closed state.

[00127] HIV-1 Env glycoprotein opening transitions can be induced by increasing temperature

[00128] The TR, T-jump SAXS experiments perturb the conformational equilibrium via a rapid (-250 ns) infrared laser induced system temperature jump. It is therefore important to determine whether the transition of interest will be sufficiently sensitive to changes in temperature over the anticipated range. For these experiments ,we selected the non-stabilized CH505 SOSIP due to its considerable lability. To determine the effects of increasing temperature on the conformational dynamics we measured 17B, 19B and PGT145 binding 25°C, 37.5°C, 45°C, and 50°C. Elevated temperatures increased 17B and 19B binding while reducing PGT145 binding, consistent with increases in the population of open or open like states (FIG. 11C) The largest differences in binding occurred at 45°C and 50°C indicating more particles transition to an open-like state as the temperature is increased. We next asked whether SAXS would capture structural changes associated with increased temperature. We measured static SAXS scattering profiles for the CH505 SOSIP at 25°C, 35°C. 40°C, 44°C, and 50°C (FIG. 16A). The difference profiles display the same features as observed in the difference profiles for CH505 and BG505 SOSIPs between q=0.01-0.05 A’¹ and at 0.07 A’¹ (FIG. 11D). These results demonstrate that the conformational transitions in CH505 SOSIP are sensitive to temperature and can be measured using SAXS.

[00129] Time resolved, temperature-jump SAXS reveals time-dependent changes in CH505 SOSIP SAXS Profiles

[00130] We next examined the CH505 SOSIP response to rapid system heating using TR, T-jump SAXS. We first optimized the initial, equilibrium temperature of our system. Based on our previous temperature dependent binding experiments, we initiated temperatures jumps from 40°C, 42°C, 44°C, and 46°C with a probe delay of 10 ps and 1ms finding that the difference signal in the region of interest (0.02-0. 10 A'¹) was maximal at 44°C. We next determined the extent of laser induced heating of the system when jumping from 44°C. Linear regression analysis (FIG. 18B) of the static SAXS difference profiles water ring maxima (1.5-2.5 A'¹) at A2°C, A5°C, A6°C, A8°C, and Al 1°C (Fig. 18A) indicated our system temperature jump was ~A6°C leading to a final perturbed system temperature of 50°C. Next, to probe the CH505 SOSIP conformation at different times post heating, we measured scattering at delay times of 500 ns, 1.5 ps, 3 ps, 5 ps, 10 ps, 50 ps, 100 ps, 500 ps, 1 ms, 10 ms, and 100 ms (FIG. 12A, FIG. 17B). The scattering difference cune measured at a post laser temperature jump time of 500 ns shows a prominent negative peak between 0.02 and 0.06 A'¹ (FIG. 17E) indicative of a process than occurs faster than our measurement dead time that may correspond to solvent shell and/or glycan rearrangements. This feature becomes increasingly prominent at greater time delays up to 1 ms with a marked increase between 0.02 and 0.03 A'¹ between 1.5 ps and 3 ps. Scattering at these low angles reports on the largest scale changes in the system, suggestive of a particle whose radius is increasing. A second signal at q = 0.07 A^-1 becomes more prominent as the delay time is increased (FIG. 12A). Both the lowest scattering angle difference and 0.07 A'¹ features are consistent with the static temperature difference SAXS curves (FIG. 11B, D). At longer time delays of 10ms and 100ms. a period over which the water ring curve maxima indicates the system temperature is dropping (FIG. 18B), these difference features begin to shift toward zero (FIG. 12A), indicating the Env relaxes back to its initial conformation. Together, these results demonstrate the HIV-1 Env conformation exhibits transitions on the microsecond timescale.

[00131] Env Opening Proceeds Through a Rapidly Forming Intermediate

[00132] We next determined the number of distinct states observed and their exchange kinetics. For this, we first extracted component curves from the set of difference curves using singular value decomposition (SVD). This method splits the data into matrices of component curves, or left vectors, singular values, and contributions, or right vectors, that when multiplied, return the original curve. This acts to remove noise from the data and to separate signals into distinct processes. Here, we focused on difference curves between 1.5 ps and 1 ms as these report primarily on structural transitions prior to cooling of the sample. The results indicated that there are two primary’ components in the time resolved SAXS difference profiles (FIG. 19A). The first left vector is comprised of a negative peak between 0.02 and 0.06 A’¹ with a minor positive peak at 0.07 A'¹ (FIG. 12B). The second left vector shows features matching an inversion of the BG505 vs. CH505 difference curve (Fig. 12B). The right vectors report on the relative contributions of each component to each individual scattering curve and show that the first component transitions at both shorter and longer time delays while the second component transitions only at longer time delays (FIG. 12E). Both the left vectors (FIG. 19B) and right vectors (Fig. 19C) for SVD components 3-8 fluctuate randomly about zero, indicating that these components do not contribute significantly to the time resolved SAXS signal. We next fit kinetic models to each of the first two distinct component right vectors. A double exponential fit to the first component yielding inverse rate constants of 4.3±0.4 ps and 574.7±0.4 ps. A double exponential fit to the second right vector yielded an inverse rate constant of 3±2 ps and 600.0±0.1 ps. The fast rate constant determined for the second right vector is not a good fit, however the slow rate constant fits well and is consistent with the slower process in the first component. We next determined rates from a double exponential fit to the area under each TR, T-Jump difference profile between 0.02 and 0.1 A'¹ (Fig. 12G). This yielded inverse rate constants of ~11 ps and the other with a rate constant of ~800ps both of which are consistent with the values from SVD. These models indicate that there is a rapid transition to a relatively long-lived intermediate conformation followed by a second, slower transition.

[00133] Singular value decomposition does not necessarily decompose data into physically meaningful components (71). A recently developed algorithm, termed REGALS. deconvolves SAXS datasets by applying experimentally determined restraints to SVD deconvolutions so that the components are physically realistic. We performed a REGALS deconvolution on TR, T-Jump SAXS difference profiles to identify individual component scattering profiles in the CH505 SOSIP time resolved SAXS data (FIG. 12C). We included two components as identified in both the SVD and area under the curve analyses with maximum dimensions of 154A and 173 A respectively, based on the dimensions of Env structure models and iterative optimization (FIG. 20). Fitting of the data yielded and overall X² value near one (FIG. 19D) with each of the components displaying similarities to their respective SVD components (Fig. 12B). The REGALS concentration estimates indicate that component 2 is gradually increasing at each time delay whereas component 1 decreases in concentration (FIG. 12F), again consistent with the results of SVD analysis. In addition to splitting the scattering difference curves into distinct components, REGALS returns pair distance distributions describing component particle atom-atom distances differences (FIG. 12D). The distance distribution for component 2 indicated structural changes occur on a relatively large range up to -120 A, with marked reductions in pairwise distances between 25 A and 75 A and concomitant increases between 80A and 120A. Distance changes for component 1 were over a smaller range of -60 A (FIG. 12D). The concentration of component 2 begins to increase at longer time delays while component 1 decreases at early and long time delays, a feature again common with the SVD analysis (FIG. 12B). These results are consistent with a model of an early intermediate structural state giving way to a more open like structural state after a delay of nearly one millisecond.

[00134] Rapidly forming Env opening intermediate consistent with a single protomer open structure

[00135] The time resolved SAXS difference profiles for CH505 Env SOSIP limit direct identification of specific structural states corresponding to the differing component scattering difference curves. Previous smFRET studies have shown that an asymmetric intermediate connects the closed and open configurations (67-70). Transitions from the ground state to this asymmetric state often occur within the dead time of the measure (-1-3 ms) which is consistent with the ~800ps timescale for the slow process measured here (FIG. 12G). The asymmetric state is characterized by a single protomer open state with tw o protomers in a distinct configuration. With the final state here likely corresponding to this state, we next examined the structural state of the fast-forming intermediate. We first asked w hether difference SAXS measures in the range of our signal can distinguish between open and open occluded states. Examination of difference curves generated from Man9 glycosylated model based theoretical profiles suggests we can (FIG. 13C). We next examined difference curves (FIG. 13A) determined from theoretical profiles (FIG. 20E) generated using models of CH505 SOSIP with (1) three protomers closed (3Closed) (FIG. 20A), (2) 1 protomer in open occluded (1 Occluded) (FIG. 20B), and (3) 3 protomers in open occluded (3 Occluded) (FIG. 20D). Difference curves between closed and open states could effectively capture observations in the experimental difference curves (FIG. 13A). Further, theoretical distance distribution difference curves (FIG. 13B) of the closed vs. 3 open occluded and closed vs. one open occluded models match the experimental P(r) functions for REGALS components 2 and 1, respectively (FIG. 12C). These results indicate the fast-forming intermediate structure is consistent with a model in which a single protomer gpl20 has changed position.

[00136] The HIV-1 Env is a heavily glycosylated protein with a dense network of conformationally dynamic interactions. We therefore asked, as an alternative to gpl20 motion, whether changes in the glycan shield could explain the data. We first compared nonglycosylated and Man9 glycosylated models. Examination of SVD left vectors extracted from theoretical scattering curves indicated the overall features between the two are similar with shifts in the scattering vector position of the peaks that are related to changes in particle size. Next, we asked whether glycan rearrangements arising from dyanmics on a single state could yield the experimentally observed difference profiles.

[00137] Finally, with the fast-forming intermediate likely corresponding to a change in apex contacts through movement of the gp!20, we asked whether this movement involves changes in the apex conformation. Isolated gpl20 domains in solution are known to expose the bridging sheet epitope targeted by 17B as well as the V3 loop. Absent timer apex contacts, a closed state gpl20 should therefore spontaneously sample this exposed state. We reasoned that, if V1/V2 and V3 exposure are involved in the fast transition, these transitions can occur in a free gpI20 within the measured intermediate timescale. We collected two- hundred and fifity independent 5-p.s molecular dynamics simulation trajectories run at 50°C to determine whether these transitions are sufficiently fast to be involved in the early- intermediate formation. The root mean square deviation (RMSD) of backbone atoms in the gp!20 core |3-sheets was markedly smaller than both V3 and V1/V2 (FIG. 13E). Fraying of the V I/V2 and V3 loops that form apex contacts in the closed state trimer was observed. Temporary release of VI loop contacts with the GDIR motif were observed in several simulations. However, we did not observe V1/V2 or V3 release consistent with epitope exposure in any of replicates (FIG. 13E). This indicates that the rapidly forming intermediate does not involve major rearrangements in the allosteric machinery that exposes these structural elements.

[00138] Interprotomer Disulfide Bonds Stabilize the Closed Env Trimer and Improve Neutralization Profiles [00139] The TR, T-Jump SAXS results indicate an intermediate structural state precedes movements to a more open configuration. A recent macaque immunization study found that, despite immunogen stabilization in a prefusion closed configuration, antibodies targeting open-occluded states were induced (43). Our modeling of the SAXS data indicates a single protomer moves first and at a rate inconsistent with major rearrangements in the allosteric machinery. Most stabilizing designs focus on disabling allosteric rearrangements and improving gp!20 to gp41 interactions. The induction of apex binding, open-occluded antibodies in vaccination with pre-fusion stabilized trimers and the presence of an intermediate consistent with this state here indicates stabilization betw een apex contacts is can eliminate open-occluded state antibody interactions.

[00140] We therefore identified tw o possible sites for introduction of a disulfide staple between gpl20 protomers at V1/V2 contact regions in a CH505 SOSIP trimer design (CH505.M5.G458Y). In some embodiments, this includes a 127C-167C mutant. In some embodiments, these were further stabilized using the previously reported F14 and SOSIP 2P mutations to improve expression and folding. The 127C-167C mutant, which displayed a marked improvement in thermal denaturation inflection temperature (Ti +7.6 °C; FIG. 14D). Examining disulfide formation in the purified material by SDS-PAGE indicated a portion of the expressed Env SOSIP may not have properly formed the inter-protomer disulfide. Consistent with this observation, binding of 19B, an exposed V3 targeting antibody, and PGT151. a trimer specific broadly neutralizing antibody, by BLI. indicated the purified trimer displayed a more open configuration (FIG. 14). Further, addition of CD4-Ig increased exposure of the 19B epitope and induced 17B epitope exposure. A clear closed state trimeric configuration w as identified in a negative-stain electron microscopy 3D reconstruction (FIG.

14A)

[00141] We therefore asked whether specific tertiary structural difference accompanied differences in apex disulfide formation. We identified two distinct structural states via single particle cryo-EM including a closed and a partially open state. The closed state configuration shows rearrangements in the V I/V2 apex inter-protomer contact loop relative to the parent, non-disulfide linked design (FIG. 14C) that included a rearrangement that introduces an inter-protomer salt bridge between R194 and E164 (FIG. 14B). These rearrangements are consistent with formation of the inter-protomer disulfide link and indicate the aberrant disulfide formation results in the partially open state. We therefore further purified the sample by negative selection using the V3 loop targeting 3074 antibody. This resulted in the elimination of the non-inter-protomer linked bands in SDS-PAGE, elimination of 19B binding, enhancement of PGT151 binding, and elimination of CD4-Ig triggering (FIGs. 14, 21A-D)

[00142] We next asked whether open occluded state antibody binding is eliminated in the inter-protomer disulfide linked trimers. We recently identified an open occluded state binding antibody in a non-human primate vaccination trial. Comparing binding of this antibody to the CH505 transmitted founder SOSIP using in the TR, T-jump SAXS and the parent design indicated the inter-protomer disulfide linked trimer effectively blocks binding of this antibody. Together, these results show that disulfide linkages at the trimer apex between protomers can effectively eliminate movements to an open-occluded, intermediate state.

[00143] Additional data are show n in FIG. 21A-F and in FIG. 22A-F.

[00144] These data indicated that, in Env opening, an initial, rapid transition involves an Env movement into a one protomer open occluded conformation along with changes in the glycan shield structure. This rapidly forming, one protomer open occluded intermediate Env is relatively long-lived. The second transition is slower and involves dislocation of the V1/V2 and V3 elements, resulting in transition of Env to a one protomer open conformation, with further rearrangements in glycan shields. MD simulations showing that neither the V 1/V 2 nor the V3 allosteric elements release on the 5ps timescales support the interpretation that Env transitions to a 1 open protomer conformation through a 1 protomer open occluded conformation during the initial stages of Env opening

[00145] Targeted design of Env SOSIP immunogens to prevent formation of the one open occluded structure showed improved closed conformation stability and neutralization profiles, further supporting the interpretation that HIV-1 Env opens through a rapidly forming, unstable one protomer open occluded transition intermediate before transition to a more stable one protomer open conformation. These results demonstrate the importance of stabilizing VI /V2 interprotomer contacts at the trimer apex for stabilizing with closed Env conformation.

[00146] References Cited in this Example

[00147] 43. Z. Yang et al., Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer. Nat Commun 13, 732 (2022).

[00148] 67. J. B. Munro et al., Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. Science 346, 759-763 (2014). [00149] 68. J. B. Munro, W. Mothes, Structure and Dynamics of the Native HIV-1 Env Trimer. J Virol 89. 5752-5755 (2015).

[00150] 69. M. Lu et al.. Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET. Nature 568, 415-419 (2019).

[00151] 70. X. Ma et al., HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations. Elife 7 (2018).

[00152] 71. S. P. Meisburger. D. Xu. N. Ando. REGALS: a general method to deconvolve X-ray scattering data from evolving mixtures. lUCrJ 8, 225-237 (2021).

[00153] EXAMPLE 3 - Sequences of Modified Env Proteins and Nucleotide Sequences Encoding the Proteins

[00154] Disclosed herein are modified HIV Env proteins that are modified so that, when the proteins are part of an Env trimer, the capability of the trimer in a closed or prefusion state, to form one of the structural intermediate states described herein and/or the open state, is decreased. In some embodiments, modifications to HIV envelope proteins to accomplish this can stabilize VI ZV2 interprotomer contacts at an apex of an Env trimer. In some embodiments, the modifications can be mutations in Env proteins. In some embodiments, the mutations link protomers in an Env trimer.

[00155] In some embodiments, a modified envelope protein is designated:

[00156] CH505M5chim.6R.SOSIP.664v4. 1 G458Y N197D F14 V127C D167C L568P T569P (also indicated as “HV1303056_Wpro”).

[00157] In this designation:

[00158] “CH505” indicates the virus isolate from an HIV- 1 -infected individual from which this Env sequence derives;

[00159] “M5” indicates a design mutation, N279K;

[00160] “chim” indicates this Env is chimeric with gpl20 from the CH505 sequence and gp41 from a different isolate, BG505;

[00161] “ 6R” is a modification know n in the art;

[00162] “SOSIP” indicates a stabilizing gp!20 to gp41 disulfide (SOS) and HR1 helix breaking I to P mutation (IP);

[00163] “664” indicates a HXB2 based residue number at which the Env sequence is truncated. It removes the membrane proximal external region, transmembrane domain, and c- terminal domain;

[00164] “v4.1” indicates mutations are shown previously to stabilize the Env ectodomain; [00165] "G45 Y." “N197D,” “F14,” L568P,” and “T569P” are other mutations. G458Y and N197D were previously designed to enhance affinity for CH235 antibodies. F14 and L568P+T569P are additional stabilization mutations; and

[00166] “V127C D167C” is an example mutation described herein that retard the capability of an Env trimer in a closed or prefusion state, to form one of the structural intermediate states described herein and/or the open state. In some embodiments, the V127C D167C mutations can lock together an apex of an Env trimer.

[00167] In some embodiments, a modified envelope protein is designated:

[00168] CH505M5chim.6R.SOSIP.664v4.1_G458Y_N197D_F14_V127C_D167C_L568P

T569P (HV1303056_Wpro)

[00169] Example nucleotide sequence encoding the above-described modified Env (SEQ

ID NO: X):

[00170] GTCGACAAGCTTcccgggccaccATGCCCATGGGCTCCCTGCAGCCCCTGGC CACCCTGTACCTGCTGGGCATGCTGGTGGCCTCCGTGCTGGCCGCCGAGAACCTG TGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCAAGACCACCCTG TTCTGCGCCTCCGACGCCAAGGCCTACGAGAAGAAGGTGCACAACATATGGGCC ACCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATGGTGCTGAAGAAC GTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTGGACCAGATGCACGAG GACGTGATCTCCCTGTGGGACCAGTCCCTGAAGCCCTGCGTGAAGCTGACCCCCC TGTGCTGCACCCTGAACTGCACCAACGCCACCGCCTCCAACTCCTCCATCATCGA GGGCATGAAGAACTGCTCCTTCAACATCACCACCGAGCTGCGCTGCAAGCGCGA GAAGAAGAACGCCCTGTTCTACAAGCTGGACATCGTGCAGCTGGACGGCAACTC CTCCCAGTACCGCCTGATCAACTGCGACACCTCCGTGATCACCCAGGTCTGCCCC AAGCTGTCCTTCGACCCCATCCCCATCCACTACTGCGCCCCCGCCGGCTACGCCA TCCTGAAGTGCAACAACAAGACCTTCACCGGCACCGGCCCCTGCAACAACGTGT CCACCGTGCAGTGCACCCACGGCATCAAGCCCGTGCTGTCCACCCAGCTGCTGCT GAACGGCTCCCTGGCCGAGGGCGAGATCATCATCCGCTCCGAGAACATCACCAA GAACGTGAAGACCATCATCGTGCACCTGAACGAGTCCGTGAAGATCGAGTGCAC CCGCCCCAACAACAAGACCCGCACCTCCATCCGCATCGGCCCCGGCCAGTGGTTC TACGCCACCGGCCAGGTGATCGGCGACATCCGCGAGGCCTACTGCAACATCAAC GAGTCCAAGTGGAACGAGACCCTGCAGCGCGTGTCCAAGAAGCTGAAGGAGTAC TTCCCCCACAAGAACATCACCTTCCAGCCCTCCTCCGGCGGCGACCTGGAGATCA CCACCCACTCCTTCAACTGCGGCGGCGAGTTCTTCTACTGCAACACCTCCTCCCT GTTCAACCGCACCTACATGGCCAACTCCACCGACATGGCCAACTCCACCGAGAC CAACTCCACCCGCACCATCACCATCCACTGCCGCATCAAGCAGATCATCAACATG TGGCAGGAGGTGGGCCGCGCCATGTACGCCCCCCCCATCGCCGGCAACATCACC TGCATCTCCAACATCACCGGCCTGCTGCTGACCCGCGACtaCGGCAAGAACAACA CCGAGACCTTCCGCCCCGGCGGCGGCAACATGAAGGACAACTGGCGCTCCGAGC TGTACAAGTACAAGGTGGTGAAGATCGAGCCCCTGGGCGTGGCCCCCACCCGCT GCAAGCGCCGCGTGGTGGGCCGCCGCCGCCGCCGCCGCGCCGTGGGCATCGGCG CCGTGTTCCTGGGCTTCCTGGGCGCCGCCGGCTCCACCATGGGCGCCGCCTCCAT GACCCTGACCGTGCAGGCCCGCAACCTGCTGTCCGGCATCGTGCAGCAGCAGTC CAACCTGCTGCGCGCCCCCGAGGCCCAGCAGCACCTGCTGAAGCCGCCCGTGTG GGGCATCAAGCAGCTGCAGGCCCGCGTGCTGGCCGTGGAGCGCTACCTGCGCGA CCAGCAGCTGCTGGGCATCTGGGGCTGCTCCGGCAAGCTGATCTGCTGCACCAAC

GTGCCCTGGAACTCCTCCTGGTCCAACCGCAACCTGTCCGAGATCTGGGACAACA

TGACCTGGCTGCAGTGGGACAAGGAGATCTCCAACTACACCCAGATCATCTACG

GCCTGCTGGAGGAGTCCCAGAACCAGCAGGAGAAGAACGAGCAGGACCTGCTG

GCCCTGGACTAGGGATCC

[00171] Example amino acid sequence of the above-described modified Env (SEQ ID NO:

X):

[00172] ROASRATMPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKE

AKTTLFCASDAKAYEKKVHNIWATHACVPTDPNPQEMVLKNVTENFNMWKNDMV

DQMHEDVISLWDQSLKPCVKLTPLCCTLNCTNATASNSSIIEGMKNCSFNITTELRCK

REKKNALFYKLDIVOLDGNSSOYRLINCDTSVITQVCPKLSFDPIPIHYCAPAGYAILK

CNNKTFTGTGPCNNVSTVOCTHGIKPVLSTQLLLNGSLAEGEIIIRSENITKNVKTIIVH

LNESVKIECTRPNNKTRTSIRIGPGOWFYATGOVIGDIREAYCNINESKWNETLQRVS

KKLKEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANS

TETNSTRTITIHCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTET

FRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFL

GFLGAAGSTMGAASMTLTVQARNLLSGIVQOQSNLLRAPEAQQHLLKPPVWGIKQL

QARVLAVERYLRDQOLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQW

DKEISNYTQIIYGLLEESONOQEKNEQDLLALD-GS

[00173] In some embodiments, a modified envelope protein is designated:

[00174] CH505M5chim.6R. SOSIP.664v4.1_G458Y_N 197D_F 14_N 197C_E 164C

(HV 1303057).

[00175] Example nucleotide sequence encoding the above-described modified Env (SEQ

ID NO: X):

[00176] GTCGACAAGCTTcccgggccaccATGCCCATGGGCTCCCTGCAGCCCCTGGC

CACCCTGTACCTGCTGGGCATGCTGGTGGCCTCCGTGCTGGCCGCCGAGAACCTG

TGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCAAGACCACCCTG

TTCTGCGCCTCCGACGCCAAGGCCTACGAGAAGAAGGTGCACAACATATGGGCC

ACCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATGGTGCTGAAGAAC

GTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTGGACCAGATGCACGAG

GACGTGATCTCCCTGTGGGACCAGTCCCTGAAGCCCTGCGTGAAGCTGACCCCCC

TGTGCGTGACCCTGAACTGCACCAACGCCACCGCCTCCAACTCCTCCATCATCGA

GGGCATGAAGAACTGCTCCTTCAACATCACCACCTGCCTGCGCGACAAGCGCGA

GAAGAAGAACGCCCTGTTCTACAAGCTGGACATCGTGCAGCTGGACGGCAACTC

CTCCCAGTACCGCCTGATCAACTGCTGCACCTCCGTGATCACCCAGGTCTGCCCC

AAGCTGTCCTTCGACCCCATCCCCATCCACTACTGCGCCCCCGCCGGCTACGCCA

TCCTGAAGTGCAACAACAAGACCTTCACCGGCACCGGCCCCTGCAACAACGTGT

CCACCGTGCAGTGCACCCACGGCATCAAGCCCGTGCTGTCCACCCAGCTGCTGCT

GAACGGCTCCCTGGCCGAGGGCGAGATCATCATCCGCTCCGAGAACATCACCAA

GAACGTGAAGACCATCATCGTGCACCTGAACGAGTCCGTGAAGATCGAGTGCAC

CCGCCCCAACAACAAGACCCGCACCTCCATCCGCATCGGCCCCGGCCAGTGGTTC

TACGCCACCGGCCAGGTGATCGGCGACATCCGCGAGGCCTACTGCAACATCAAC

GAGTCCAAGTGGAACGAGACCCTGCAGCGCGTGTCCAAGAAGCTGAAGGAGTAC

TTCCCCCACAAGAACATCACCTTCCAGCCCTCCTCCGGCGGCGACCTGGAGATCA

CCACCCACTCCTTCAACTGCGGCGGCGAGTTCTTCTACTGCAACACCTCCTCCCT

GTTCAACCGCACCTACATGGCCAACTCCACCGACATGGCCAACTCCACCGAGAC CAACTCCACCCGCACCATCACCATCCACTGCCGCATCAAGCAGATCATCAACATG

TGGCAGGAGGTGGGCCGCGCCATGTACGCCCCCCCCATCGCCGGCAACATCACC

TGCATCTCCAACATCACCGGCCTGCTGCTGACCCGCGACtaCGGCAAGAACAACA

CCGAGACCTTCCGCCCCGGCGGCGGCAACATGAAGGACAACTGGCGCTCCGAGC

TGTACAAGTACAAGGTGGTGAAGATCGAGCCCCTGGGCGTGGCCCCCACCCGCT

GCAAGCGCCGCGTGGTGGGCCGCCGCCGCCGCCGCCGCGCCGTGGGCATCGGCG

CCGTGTTCCTGGGCTTCCTGGGCGCCGCCGGCTCCACCATGGGCGCCGCCTCCAT

GACCCTGACCGTGCAGGCCCGCAACCTGCTGTCCGGCATCGTGCAGCAGCAGTC

CAACCTGCTGCGCGCCCCCGAGGCCCAGCAGCACCTGCTGAAGCTGACCGTGTG

GGGCATCAAGCAGCTGCAGGCCCGCGTGCTGGCCGTGGAGCGCTACCTGCGCGA

CCAGCAGCTGCTGGGCATCTGGGGCTGCTCCGGCAAGCTGATCTGCTGCACCAAC

GTGCCCTGGAACTCCTCCTGGTCCAACCGCAACCTGTCCGAGATCTGGGACAACA

TGACCTGGCTGCAGTGGGACAAGGAGATCTCCAACTACACCCAGATCATCTACG

GCCTGCTGGAGGAGTCCCAGAACCAGCAGGAGAAGAACGAGCAGGACCTGCTG

GCCCTGGACTAGGGATCC

[00177] Example amino acid sequence of the above-described modified Env (SEQ ID NO:

X):

[00178] ROASRATMPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKE

AKTTLFCASDAKAYEKKVHNIWATHACVPTDPNPQEMVLKNVTENFNMWKNDMV

DQMHEDVISLWDQSLKPCVKLTPLCVTLNCTNATASNSSIIEGMKNCSFNITTCLRDK

REKKNALFYKLDIVQLDGNSSOYRLINCCTSVITQVCPKLSFDPIPIHYCAPAGYAILK

CNNKTFTGTGPCNNVSTVOCTHGIKPVLSTQLLLNGSLAEGEIIIRSENITKNVKTIIVH

LNESVKIECTRPNNKTRTSIRIGPGOWFYATGOVIGDIREAYCNINESKWNETLQRVS

KKLKEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANS

TETNSTRTITIHCRIKQIINMWOEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTET

FRPGGGNMKDNWRSELYKYKVVKJEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFL

GFLGAAGSTMGAASMTLTVOARNLLSGIVOOOSNLLRAPEAOQHLLKLTVWGIKOL

QARVLAVERYLRDOOLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQW

DKEISNYTQIIYGLLEESONOQEKNEQDLLALD-GS

[00179] The table below indicates other example modified Env proteins, and example nucleotide sequences encoding the proteins. These proteins were designed such that, when the proteins are part of an Env trimer, the capability of the trimer in a closed or prefusion state, to form one of the structural intermediate states described herein and/or the open state, is decreased.

[00180] Example amino acid sequences of the Env proteins indicated in Table 1 and example nucleotide sequences encoding the Env proteins are below.

[00181] HV1301379 V127C D167C (SEQ ID NO: X):

[00182] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATG

CCTATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCG CAAGTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGT

CTGGAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAG ACCGAAAAGCACAATGTCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAAC

CCCCAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAAC AATATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTG

AAGCCCTGCGTCAAACTGACTCCTCTGTGCTGCACCCTGCAGTGTACCAATGTCA CAAACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACA

TGACCACAGAACTGAGATGCAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGC TGGATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACA

AGGAATACAGACTGATCAATTGCAACACTTCCGCCATTACCCAGGCTTGTCCTAA AGTGTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCC

TGAAGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTAC AGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACACAGCTGCTGCTGAAT

GGAAGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAAT GCCAAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGA

CCCAACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACG CTACAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGG

CAACCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCG GGAATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCA

CTACCCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCT GTTTAATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGG

TCAAACGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGC AGCGGATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGT GAGCAATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTAC AACTGAAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGA GCTGTACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAG ATGCAAGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTG GGGCCGTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTC AATGACTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCA GTCCAACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCTGACCGT GTGGGGCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAG AGACCAGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACA AATGTGCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACA ATATGACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTA CGGCCTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCT GGCACTGGATTGAGGATCC [00183] »HV1301379_V127C_D167C_aaSequence (SEQ ID NO: X):

[00184] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETT

LFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHT DIISLWDQSLKPCVKLTPLCCTLQCTNVTNNITDDMRGELKNCSFNMTTELRCKKQK VYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPA GFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITN NAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATW NETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRD GGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRR AVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIW DNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

[00185] >HV1301379_N197C_E164C (SEQ ID NO: X):

[00186] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATG

CCTATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCG CAAGTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGT CTGGAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAG ACCGAAAAGCACAATGTCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAAC CCCCAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAAC AATATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTG AAGCCCTGCGTCAAACTGACTCCTCTGTGCGTGACCCTGCAGTGTACCAATGTCA CAAACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACA TGACCACATGCCTGAGAGACAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGC TGGATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACA AGGAATACAGACTGATCAATTGCTGCACTTCCGCCATTACCCAGGCTTGTCCTAA AGTGTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCC TGAAGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTAC AGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACACAGCTGCTGCTGAAT GGAAGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAAT GCCAAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGA CCCAACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACG CTACAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGG CAACCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCG GGAATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCA CTACCCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCT GTTTAATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGG TCAAACGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGC AGCGGATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGT GAGCAATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTAC AACTGAAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGA GCTGTACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAG ATGCAAGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTG GGGCCGTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTC AATGACTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCA GTCCAACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCTGACCGT GTGGGGCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAG

AGACCAGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACA AATGTGCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACA

ATATGACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTA

CGGCCTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCT GGCACTGGATTGAGGATCC

[00187] »HV1301379_N197C_E164C_aaSequence (SEQ ID NO: X):

[00188] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC ASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII

SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTCLRDKKQKVY SLFYRLDVVQINENQGNRSNNSNKEYRLINCCTSAITQACPKVSFEPIPIHYCAPAGFA ILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAK NILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETL GKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTS VQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGS TNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAV GIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLT VWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDN MTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

[00189] >HV1301379_V127C_D167C_Wpro (SEQ ID NO: X):

[00190] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCCT ATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCAA GTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCTG GAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGACC GAAAAGCACAATGTCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCCC CAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAAT ATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAAG CCCTGCGTCAAACTGACTCCTCTGTGCTGCACCCTGCAGTGTACCAATGTCACAA ACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATGA CCACAGAACTGAGATGCAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTGG ATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAGG AATACAGACTGATCAATTGCAACACTTCCGCCATTACCCAGGCTTGTCCTAAAGT GTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTGA AGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAGT CCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACACAGCTGCTGCTGAATGGA AGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGCC AAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC

AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTGGGGCC GTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTCAATGA

CTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCAGTCCA

ACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCCGCCCGTGTGGG

GCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAGAGACC

AGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACAAATGT

GCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACAATATG

ACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTACGGC

CTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCTGGC

ACTGGATTGAGGATCC

[00191] »HV1301379_V127C_D167C_Wpro_aaSequence (SEQ ID NO: X):

[00192] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC

ASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII

SLWDQSLKPCVKLTPLCCTLQCTNVTNNITDDMRGELKNCSFNMTTELRCKKQKVY

SLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFA

ILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAK

NILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETL

GKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTS

VQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGS

TNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAV

GIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKPP

VWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDN

MTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

[00193] >HV1301379_N197C_E164C_Wpro (SEQ ID NO: X):

[00194] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCCT

ATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCAA

GTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCTG

GAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGACC

GAAAAGCACAATGTCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCCC

CAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAAT

ATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAAG

CCCTGCGTCAAACTGACTCCTCTGTGCGTGACCCTGCAGTGTACCAATGTCACAA

ACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATGA

CCACATGCCTGAGAGACAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTGG

ATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAGG

AATACAGACTGATCAATTGCTGCACTTCCGCCATTACCCAGGCTTGTCCTAAAGT

GTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTGA

AGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAGT

CCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACACAGCTGCTGCTGAATGGA

AGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGCC

AAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC

AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA

CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA

CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA

ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC

CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA

ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA

CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA

ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG

AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT

ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA

AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTGGGGCC

GTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTCAATGA

CTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCAGTCCA

ACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCCGCCCGTGTGGG

GCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAGAGACC

AGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACAAATGT

GCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACAATATG

ACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTACGGC

CTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCTGGC

ACTGGATTGAGGATCC

[00195] »HV1301379_N197C_E164C_Wpro_aaSequence (SEQ ID NO: X):

[00196] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC ASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII

SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTCLRDKKQKVY SLFYRLDVVQINENQGNRSNNSNKEYRLINCCTSAITQACPKVSFEPIPIHYCAPAGFA ILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAK NILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETL GKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTS VQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGS TNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAV GIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKPP VWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDN MTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

[00197] >HV1303255_FI4 (SEQ ID NO: X):

[00198] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCCT ATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCAA GTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCTG GAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGACC GAAAAGCACAATATCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCCC CAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAAT ATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAAG CCCTGCGTCAAACTGACTCCTCTGTGCTGCACCCTGCAGTGTACCAATGTCACAA ACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATGA CCACAGAACTGAGATGCAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTGG

ATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAGG AATACAGACTGATCAATTGCAACACTTCCGCCATTACCCAGGTGTGTCCTAAACT GTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTGA AGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAGT CCAGTGTACTCACGGGATTAAGCCTGTGCTGTCTACACAGCTGCTGCTGAATGGA AGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGCC AAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG

ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTGGGGCC

GTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTCAATGA

CTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCAGTCCA ACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCTGACCGTGTGGG

GCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAGAGACC AGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACAAATGT GCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACAATATG ACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTACGGC CTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCTGGC

ACTGGATTGAGGATC C

[00199] »HV 1303255_F 14_aaSequence

[00200] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC ASDAKAYETEKHNIWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIIS

LWDQSLKPCVKLTPLCCTLQCTNVTNNITDDMRGELKNCSFNMTTELRCKKQKVYS LFYRLDVVQINENQGNRSNNSNKEYRL1NCNTSAITQVCPKLSFEPIP1HYCAPAGFA1

LKCKDKKFNGTGPCPSVSTVQCTHGIKPVLSTQLLLNGSLAEEEVMIRSENITNNAKN ILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLG KVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSV QGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGST NSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGI

GAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTV WGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDN

MTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD-GS

[00201]

[00202] >HV1303256_F14 (SEQ ID NO: X):

[00203] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCCT

ATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCAA GTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCTG GAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGACC GAAAAGCACAATATCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCCC CAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAAT

ATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAAG CCCTGCGTCAAACTGACTCCTCTGTGCGTGACCCTGCAGTGTACCAATGTCACAA ACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATGA CCACATGCCTGAGAGACAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTGG ATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAGG

AATACAGACTGATCAATTGCTGCACTTCCGCCATTACCCAGGTGTGTCCTAAACT GTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTGA

AGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAGT CCAGTGTACTCACGGGATTAAGCCTGTGCTGTCTACACAGCTGCTGCTGAATGGA AGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGCC AAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTGGGGCC GTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTCAATGA CTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCAGTCCA ACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCTGACCGTGTGGG GCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAGAGACC

AGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACAAATGT GCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACAATATG ACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTACGGC CTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCTGGC ACTGGATTGAGGATCC

[00204] »HV1303256_F14_aaSequence (SEQ ID NO: X):

[00205] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC ASDAKAYETEKHNIWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIIS

LWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTCLRDKKQKVYS LFYRLDVVQINENQGNRSNNSNKEYRLINCCTSAITQVCPKLSFEPIPIHYCAPAGFAI LKCKDKKFNGTGPCPSVSTVQCTHGIKPVLSTQLLLNGSLAEEEVMIRSENITNNAKN ILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLG KVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSV QGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGST NSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGI GAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTV WGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDN MTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD-GS

[00206]

[00207] >HV1303257_F14 (SEQ ID NO: X):

[00208] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCCT ATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCAA GTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCTG GAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGACC GAAAAGCACAATATCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCCC CAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAAT ATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAAG CCCTGCGTCAAACTGACTCCTCTGTGCTGCACCCTGCAGTGTACCAATGTCACAA ACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATGA CCACAGAACTGAGATGCAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTGG

ATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAGG

AATACAGACTGATCAATTGCAACACTTCCGCCATTACCCAGGTGTGTCCTAAACT

GTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTGA

AGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAGT

CCAGTGTACTCACGGGATTAAGCCTGTGCTGTCTACACAGCTGCTGCTGAATGGA

AGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGCC

AAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC

AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA

CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA

CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA

ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC

CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA

ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA

CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG

ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA

ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG

AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT

ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA

AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTGGGGCC

GTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTCAATGA

CTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCAGTCCA

ACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCCGCCCGTGTGGG

GCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAGAGACC

AGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACAAATGT

GCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACAATATG

ACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTACGGC

CTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCTGGC

ACTGGATTGAGGATCC

[00209] »HV1 303257 F14_aaSequence (SEQ ID NO: X):

[00210] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKD AETT

LFCASDAKAYETEKHNIWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHT

DIISLWDQSLKPCVKLTPLCCTLQCTNVTNNITDDMRGELKNCSFNMTTELRCKKQK

VYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQVCPKLSFEPIPIHYCAPAG

FAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVLSTQLLLNGSLAEEEVMIRSENITNN

AKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWN

ETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWIS

NTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRD

GGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRR

AVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK

PPVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIW

DNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD-GS

[00211]

[00212] >HV1303258_F14 (SEQ ID NO: X):

[00213] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCCT

ATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCAA

GTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCTG

GAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGACC GAAAAGCACAATATCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCCC CAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAAT ATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAAG CCCTGCGTCAAACTGACTCCTCTGTGCGTGACCCTGCAGTGTACCAATGTCACAA ACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATGA

CCACATGCCTGAGAGACAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTGG ATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAGG AATACAGACTGATCAATTGCTGCACTTCCGCCATTACCCAGGTGTGTCCTAAACT GTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTGA

AGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAGT CCAGTGTACTCACGGGATTAAGCCTGTGCTGTCTACACAGCTGCTGCTGAATGGA AGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGCC AAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA

CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA

CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA

AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTGGGGCC GTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTCAATGA CTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCAGTCCA ACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCCGCCCGTGTGGG GCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAGAGACC

AGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACAAATGT GCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACAATATG ACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTACGGC CTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCTGGC ACTGGATTGAGGATCC

[00214] »HV1303258_F14_aaSequence (SEQ ID NO: X):

[00215] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC ASDAKAYETEKHNIWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIIS

LWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTCLRDKKQKVYS LFYRLDVVQINENQGNRSNNSNKEYRLINCCTSAITQVCPKLSFEPIPIHYCAPAGFAI

LKCKDKKFNGTGPCPSVSTVQCTHGIKPVLSTQLLLNGSLAEEEVMIRSENITNNAKN ILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLG KVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSV QGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGST NSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGI

GAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKPPVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMT WLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD-GS EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.

Claims

CLAIMS What is claimed:

1. A modified Env protein, comprising a modification that, when the protein is part of an Env trimer, decreases capability' of the Env trimer in a closed or prefusion state, to form a one protomer open occluded structure, a one protomer open structure, an open state and/or a postfusion state.

2. The modified Env protein of claim 1, wherein the modification stabilizes VI IN 2 interprotomer contacts at an apex of the Env trimer

3. The modified Env protein of claim 1, wherein the mutation comprises amino acids that form disulfide bonds or salt bridges between protomers in the Env trimer, amino acid linkers that stabilize a backbone fold, or combinations thereof.

4. A modified Env protein, comprising a mutation that links the protein with an adjacent modified Env protein when the modified Env protein forms an Env trimer.

5. The modified Env protein of claim 4, wherein the mutation is in a VI region, V2 region, V3 region, and/or 02O-P21 region of Env.

6. The modified Env protein of claim 4, wherein the mutation comprises an amino acid substitution.

7. The modified Env protein of claim 4, wherein the Env trimer comprising modified Env proteins has a stabilized apex.

8. The modified Env protein of claim 4, wherein the Env trimer has a decreased probability’ of accessing an intermediate (1 occluded open) and/or open state.

9. An Env trimer, comprising 1, 2 or 3 of the modified Env proteins of any one of claims 1-7.

10. A vaccine composition comprising the modified Env protein of any one of claims 1-8 or the Env trimer of claim 9.