Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

Mapping Intimacies ◽

10.1101/2021.07.20.453076 ◽

2021 ◽

Author(s):

James Mark Binley ◽

Emma Crooks ◽

Francisco Almanza ◽

Alessio D'Addabbo ◽

Erika Duggan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Assay ◽

Network Effect ◽

Ripple Effects ◽

Rigorous Testing ◽

Outer Domain ◽

Biophysical Analysis ◽

Standard Assay ◽

Boost Vaccine ◽

Hiv 1

HIV-1 vaccine immunofocusing strategies have the potential to induce broadly reactive nAbs. Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets of neutralizing antibodies (NAbs), the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-neutralizing antibodies, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the V2 loop's C-strand. Notably, a 167N mutation improved V2-sensitivity. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing other glycans in some cases had local and global "ripple" effects on glycan maturation and sequon occupation in the gp120 outer domain and gp41. V2 mAb CH01 selectively bound trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a N49 glycan perturbs gp41 glycans via a distal glycan network effect, increasing FP NAb sensitivity - and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Overall, we identified 7 diverse trimers with a range of sensitivities to two targets that should enable rigorous testing of immunofocusing vaccine concepts.

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Conformational Engineering of HIV-1 Env Based on Mutational Tolerance in the CD4 and PG16 Bound States

Journal of Virology ◽

10.1128/jvi.00219-19 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 3

Author(s):

Jeremiah D. Heredia ◽

Jihye Park ◽

Hannah Choi ◽

Kevin S. Gill ◽

Erik Procko

Keyword(s):

Virus Replication ◽

Conformational Changes ◽

Neutralizing Antibodies ◽

Bound States ◽

Structural Changes ◽

Electrostatic Repulsion ◽

Closed Conformation ◽

Open Conformation ◽

Outer Domain ◽

Hiv 1

ABSTRACTHIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a “closed” to “open” conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design.IMPORTANCEHIV-1 Env is dynamic and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, among other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between domains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.

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Design of a Non-glycosylated Outer Domain-derived HIV-1 gp120 Immunogen That Binds to CD4 and Induces Neutralizing Antibodies

Journal of Biological Chemistry ◽

10.1074/jbc.m110.152272 ◽

2010 ◽

Vol 285 (35) ◽

pp. 27100-27110 ◽

Cited By ~ 27

Author(s):

Sanchari Bhattacharyya ◽

Roshan Elizabeth Rajan ◽

Yalla Swarupa ◽

Ujjwal Rathore ◽

Anjali Verma ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Outer Domain ◽

Hiv 1

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Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.01762-16 ◽

2016 ◽

Vol 91 (1) ◽

Cited By ~ 14

Author(s):

Hannah M. Cheeseman ◽

Natalia J. Olejniczak ◽

Paul M. Rogers ◽

Abbey B. Evans ◽

Deborah F. L. King ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Mucosal Tissue ◽

Broadly Neutralizing Antibodies ◽

Free Virus ◽

Mucosal Tissues ◽

Outer Domain ◽

Highly Effective ◽

Tissue Models ◽

Hiv 1 ◽

Rv144 Vaccine

ABSTRACT Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection. IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue have not been defined. While bnAbs are highly effective against cell-free virus, they are not induced by current vaccine candidates. However, nnAbs, readily induced by vaccines, can trigger antibody-dependent cellular effector functions, through engagement of their Fc-gamma receptors. Fc-mediated antiviral activity has been implicated as a secondary correlate of decreased HIV-1 risk in the RV144 vaccine efficacy trial, suggesting that protection might be mediated in the absence of classical neutralization. To aid vaccine design and selection of antibodies for use in passive protection strategies, we assessed a range of bnAbs and nnAbs for their potential to block ex vivo challenge of mucosal tissues. Our data clearly indicate the superior efficacy of neutralizing antibodies in preventing mucosal acquisition of infection. These results underscore the importance of maintaining the central focus of HIV-1 vaccine research on the induction of potently neutralizing antibodies.

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Chemically Modified Peptides Based on the Membrane-Proximal External Region of the HIV-1 Envelope Induce High-Titer, Epitope-Specific Nonneutralizing Antibodies in Rabbits

Clinical and Vaccine Immunology ◽

10.1128/cvi.00320-14 ◽

2014 ◽

Vol 21 (8) ◽

pp. 1086-1093 ◽

Cited By ~ 10

Author(s):

Vincent J. Venditto ◽

Lindsay Wieczorek ◽

Sebastian Molnar ◽

Fernando Teque ◽

Gary Landucci ◽

...

Keyword(s):

Neutralizing Antibodies ◽

High Titer ◽

Neutralization Assay ◽

Vaccine Strategy ◽

Chemical Modifications ◽

External Region ◽

Membrane Proximal External Region ◽

Chemically Modified ◽

Modified Peptides ◽

Hiv 1

ABSTRACTBroadly neutralizing monoclonal antibodies (bNAbs) 2F5 and 4E10 bind to the membrane proximal external region (MPER) of gp41 and also cross-react with phospholipids. In this study, we investigated if chemical modifications on the MPER adjacent to 2F5 and 4E10 epitopes using mimetics of inflammation-associated posttranslational modifications to induce 2F5- and 4E10-like bNAbs can break tolerance. We synthesized a series of chemically modified peptides spanning the MPER. The serine, threonine, and tyrosine residues in the peptides were modified with sulfate, phosphate, or nitrate moieties and presented in liposomes for rabbit immunizations. All immunizations resulted in high antisera titers directed toward both the modified and unmodified immunogens. Tyrosine modification was observed to significantly suppress antiepitope responses. Sera with strong anti-gp140 titers were purified by affinity chromatography toward the MPER peptide and found to possess a higher affinity toward the MPER than did the bNAbs 2F5 and 4E10. Modest neutralization was observed in the H9 neutralization assay, but neutralization was not observed in the TZM-bl cell or peripheral blood mononuclear cell (PBMC) neutralization assay platforms. Although neutralizing antibodies were not induced by this approach, we conclude that chemical modifications can increase the immune responses to poorly immunogenic antigens, suggesting that chemical modification in an appropriate immunization protocol should be explored further as an HIV-1 vaccine strategy.

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The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay

International Journal of Molecular Sciences ◽

10.3390/ijms23020641 ◽

2022 ◽

Vol 23 (2) ◽

pp. 641

Author(s):

Vladimir Morozov ◽

Sylvie Lagaye ◽

Alexey Morozov

Keyword(s):

Cell Line ◽

Neutralizing Antibodies ◽

False Negative ◽

Neutralization Assay ◽

Reporter Cell Line ◽

Reporter Cell ◽

Negative Results ◽

Viral Vaccine ◽

Neutralizing Monoclonal Antibody ◽

Hiv 1

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated.

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Bacterially expressed HIV-1 gp120 outer-domain fragment immunogens with improved stability and affinity for CD4-binding site neutralizing antibodies

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005006 ◽

2018 ◽

Vol 293 (39) ◽

pp. 15002-15020 ◽

Cited By ~ 5

Author(s):

Ujjwal Rathore ◽

Mansi Purwar ◽

Venkada Subramanian Vignesh ◽

Raksha Das ◽

Aditya Arun Kumar ◽

...

Keyword(s):

Binding Site ◽

Neutralizing Antibodies ◽

Outer Domain ◽

Cd4 Binding Site ◽

Improved Stability ◽

Hiv 1

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Enhanced Exposure of the CD4-Binding Site to Neutralizing Antibodies by Structural Design of a Membrane-Anchored Human Immunodeficiency Virus Type 1 gp120 Domain

Journal of Virology ◽

10.1128/jvi.02600-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 5077-5086 ◽

Cited By ~ 40

Author(s):

Lan Wu ◽

Tongqing Zhou ◽

Zhi-yong Yang ◽

Krisha Svehla ◽

Sijy O'Dell ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Neutralizing Antibodies ◽

Outer Domain ◽

Immunodeficiency Virus ◽

Cd4 Binding Site ◽

Hiv 1

ABSTRACT The broadly neutralizing antibody immunoglobulin G1 (IgG1) b12 binds to a conformationally conserved surface on the outer domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) glycoprotein. To develop outer domain proteins (ODs) that could be recognized selectively by CD4-binding-site (CD4-BS) antibodies, membrane-anchored ODs were generated from an HIV-1 clade B virus, TA1 R3A, which was highly sensitive to neutralization by the IgG1 b12 antibody. A 231-residue fragment of gp120 (residues 252 to 482) linked to transmembrane regions from CD4 showed b12 binding comparable to that of the native Env spike as measured by flow cytometry. Truncation of the β20-β21 hairpin (residues 422 to 436 to Gly-Gly) improved overall protein expression. Replacement of the immunodominant central 20 amino acids of the V3 loop (residues 302 to 323) with a basic hexapeptide (NTRGRR) increased b12 reactivity further. Surface calculations indicated that the ratio of b12 epitope to exposed immunogenic surface in the optimized OD increased to over 30%. This OD variant [OD(GSL)(Δβ20-21)(hCD4-TM)] was recognized by b12 and another CD4-BS-reactive antibody, b13, but not by eight other CD4-BS antibodies with limited neutralization potency. Furthermore, optimized membrane-anchored OD selectively absorbed neutralizing activity from complex antisera and b12. Structurally designed membrane-anchored ODs represent candidate immunogens to elicit or to allow the detection of broadly neutralizing antibodies to the conserved site of CD4 binding on HIV-1 gp120.

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