Impact of glycan positioning on HIV-1 Env glycan shield density, function, and antibody recognition

AbstractN-glycans, which represent >50% mass of the HIV-1 envelope (Env) trimer, play important roles for virus-cell entry and immune evasion. How each glycan unit interacts to shape the Env protein-sugar complex and affects Env function is not well understood. Here, high-resolution glycomics analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation-site not only affected the Env structure at distant locations, but also had a ripple effect on Env-wide glycan processing, virus infectivity, and antibody recognition and virus neutralization. Specifically, the N262 glycan, although not located in the CD4-binding site, controlled Env binding to the CD4 receptor, affected the recognition of Env by several glycan-dependent broadly neutralizing antibodies, and altered heterogeneity of glycosylation at several sites, with N156, N160, and N448 displaying limited glycan processing. Molecular dynamic simulations visualized how specific oligosaccharide positions can move to compensate for loss of a glycan. This study demonstrates how changes in individual glycan units can alter molecular dynamics and processing of the Env-glycan shield and, consequently, Env function.

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Griffithsin Retains Anti-HIV-1 Potency with Changes in gp120 Glycosylation and Complements Broadly Neutralizing Antibodies PGT121 and PGT126

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01084-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 1

Author(s):

Kathryn Fischer ◽

Kimberly Nguyen ◽

Patricia J. LiWang

Keyword(s):

Neutralizing Antibodies ◽

Glycosylation Site ◽

Significant Loss ◽

Broadly Neutralizing Antibodies ◽

Clade B ◽

Key Factor ◽

Clade C ◽

Cd4 Binding Site ◽

Gp120 Glycosylation ◽

Hiv 1

ABSTRACT Griffithsin (Grft) is an antiviral lectin that has been shown to potently inhibit HIV-1 by binding high-mannose N-linked glycosylation sites on HIV-1 gp120. A key factor for Grft potency is glycosylation at N295 of gp120, which is directly adjacent to N332, a target glycan for an entire class of broadly neutralizing antibodies (bNAbs). Here, we unify previous work on the importance of other glycans to Grft potency against HIV-1 and Grft’s role in mediating the conformational change of gp120 by mutating nearly every glycosylation site in gp120. In addition to a significant loss of Grft activity by the removal of glycosylation at N295, glycan absence at N332 or N448 was found to have moderate effects on Grft potency. Interestingly, in the absence of N295, Grft effectiveness could be improved by a mutation that results in the glycan at N448 shifting to N446, indicating that the importance of individual glycans may be related to their effect on glycosylation density. Grft’s ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in clade B strains, not clade C strains. We further demonstrate that Grft can rescue the activity of the bNAbs PGT121 and PGT126 in the event of a loss or a shift of glycosylation at N332, where the bNAbs suffer a drastic loss of potency. Despite targeting the same region, Grft in combination with PGT121 and PGT126 produced additive effects. This indicates that Grft could be an important combinational therapeutic.

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Changes in Structure and Antigenicity of HIV-1 Env Trimers Resulting from Removal of a Conserved CD4 Binding Site-Proximal Glycan

Journal of Virology ◽

10.1128/jvi.01116-16 ◽

2016 ◽

Vol 90 (20) ◽

pp. 9224-9236 ◽

Cited By ~ 17

Author(s):

Yu Liang ◽

Miklos Guttman ◽

James A. Williams ◽

Hans Verkerke ◽

Daniel Alvarado ◽

...

Keyword(s):

Binding Site ◽

Structural Dynamics ◽

Neutralizing Antibodies ◽

Glycosylation Site ◽

Protective Mechanism ◽

Immune Recognition ◽

Simple Modification ◽

Cd4 Binding Site ◽

Monomeric Gp120 ◽

Hiv 1

ABSTRACTThe envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines.IMPORTANCEThe HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.

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Membrane Env liposomes facilitate immunization with multivalent full-length HIV spikes

Journal of Virology ◽

10.1128/jvi.00005-21 ◽

2021 ◽

Author(s):

Daniel P. Leaman ◽

Armando Stano ◽

Yajing Chen ◽

Lei Zhang ◽

Michael B. Zwick

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Development ◽

Serum Antibody ◽

Glycosylation Site ◽

Broadly Neutralizing Antibodies ◽

Vaccine Platform ◽

Antibody Recognition ◽

Correct Orientation ◽

Cd4 Binding Site ◽

Antibody Specificities

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bnAbs). Such bnAbs target HIV’s trimeric, membrane embedded envelope glycoprotein spikes, (m)Env. Soluble (s)Env trimers have been used as vaccines, but engineering sEnvs for stability, multivalency and desired antigenicity is problematic, and deletes key neutralizing epitopes on glycoprotein (gp)41 while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. MEnv trimers were fixed, purified and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter 133 nm) in correct orientation. These particles were recognized by HIV bnAbs and not non-nAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provide clues on how nAb responses may be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enable rational immunization strategies to elicit HIV bnAbs using multimerized mEnv. Importance A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane embedded envelope glycoprotein spikes (m)Env that HIV uses to enter cells. Due to HIV Env’s low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane embedded targets of therapeutic interest.

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Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core

eLife ◽

10.7554/elife.37688 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 14

Author(s):

Andrew J Borst ◽

Connor E Weidle ◽

Matthew D Gray ◽

Brandon Frenz ◽

Joost Snijder ◽

...

Keyword(s):

Neutralizing Antibodies ◽

X Ray Crystallography ◽

Antibody Recognition ◽

Immunodeficiency Virus ◽

Gp120 Core ◽

Clade C ◽

Immunogen Design ◽

Cd4 Binding Site ◽

Variable Loops ◽

Hiv 1

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1–3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.

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Structural basis for germline antibody recognition of HIV-1 immunogens

eLife ◽

10.7554/elife.13783 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 37

Author(s):

Louise Scharf ◽

Anthony P West ◽

Stuart A Sievers ◽

Courtney Chen ◽

Siduo Jiang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Affinity Maturation ◽

Structural Basis ◽

Binding Modes ◽

Broadly Neutralizing Antibodies ◽

Antibody Recognition ◽

Immunogen Design ◽

Cd4 Binding Site ◽

Modified Forms ◽

Hiv 1

Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design.

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Immunogenicity, safety, and efficacy of sequential immunizations with an SIV-based IDLV expressing CH505 Envs

npj Vaccines ◽

10.1038/s41541-020-00252-w ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Maria Blasi ◽

Donatella Negri ◽

Kevin O. Saunders ◽

Erich J. Baker ◽

Hannah Stadtler ◽

...

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Lentiviral Vector ◽

Rhesus Macaques ◽

Infected Individual ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Safety And Efficacy ◽

Env Protein ◽

Hiv 1

AbstractA preventative HIV-1 vaccine is an essential intervention needed to halt the HIV-1 pandemic. Neutralizing antibodies protect against HIV-1 infection in animal models, and thus an approach toward a protective HIV-1 vaccine is to induce broadly cross-reactive neutralizing antibodies (bnAbs). One strategy to achieve this goal is to define envelope (Env) evolution that drives bnAb development in infection and to recreate those events by vaccination. In this study, we report the immunogenicity, safety, and efficacy in rhesus macaques of an SIV-based integrase defective lentiviral vector (IDLV) expressing sequential gp140 Env immunogens derived from the CH505 HIV-1-infected individual who made the CH103 and CH235 bnAb lineages. Immunization with IDLV expressing sequential CH505 Envs induced higher magnitude and more durable binding and neutralizing antibody responses compared to protein or DNA +/− protein immunizations using the same sequential envelopes. Compared to monkeys immunized with a vector expressing Envs alone, those immunized with the combination of IDLV expressing Env and CH505 Env protein demonstrated improved durability of antibody responses at six months after the last immunization as well as lower peak viremia and better virus control following autologous SHIV-CH505 challenge. There was no evidence of vector mobilization or recombination in the immunized and challenged monkeys. Although the tested vaccines failed to induce bnAbs and to mediate significant protection following SHIV-challenge, our results show that IDLV proved safe and successful at inducing higher titer and more durable immune responses compared to other vaccine platforms.

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Autologous IgG antibodies block outgrowth of a substantial but variable fraction of viruses in the latent reservoir for HIV-1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020617117 ◽

2020 ◽

Vol 117 (50) ◽

pp. 32066-32077

Author(s):

Lynn N. Bertagnolli ◽

Joseph Varriale ◽

Sarah Sweet ◽

Jacqueline Brockhurst ◽

Francesco R. Simonetti ◽

...

Keyword(s):

T Cell Activation ◽

Neutralizing Antibodies ◽

Cell Activation ◽

Viral Evolution ◽

Treatment Interruption ◽

Latent Reservoir ◽

Env Protein ◽

Autologous Antibodies ◽

Hiv 1

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4+ T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

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Neutralization of Virus Infectivity by Antibodies: Old Problems in New Perspectives

Advances in Biology ◽

10.1155/2014/157895 ◽

2014 ◽

Vol 2014 ◽

pp. 1-24 ◽

Cited By ~ 71

Author(s):

P. J. Klasse

Keyword(s):

Cellular Immunity ◽

Neutralizing Antibodies ◽

Viral Infections ◽

Viral Proteins ◽

Virus Neutralization ◽

Virus Infectivity ◽

Viral Pathogens ◽

Enveloped Viruses ◽

Vaccine Candidates ◽

Hiv 1

Neutralizing antibodies (NAbs) can be both sufficient and necessary for protection against viral infections, although they sometimes act in concert with cellular immunity. Successful vaccines against viruses induce NAbs but vaccine candidates against some major viral pathogens, including HIV-1, have failed to induce potent and effective such responses. Theories of how antibodies neutralize virus infectivity have been formulated and experimentally tested since the 1930s; and controversies about the mechanistic and quantitative bases for neutralization have continually arisen. Soluble versions of native oligomeric viral proteins that mimic the functional targets of neutralizing antibodies now allow the measurement of the relevant affinities of NAbs. Thereby the neutralizing occupancies on virions can be estimated and related to the potency of the NAbs. Furthermore, the kinetics and stoichiometry of NAb binding can be compared with neutralizing efficacy. Recently, the fundamental discovery that the intracellular factor TRIM21 determines the degree of neutralization of adenovirus has provided new mechanistic and quantitative insights. Since TRIM21 resides in the cytoplasm, it would not affect the neutralization of enveloped viruses, but its range of activity against naked viruses will be important to uncover. These developments bring together the old problems of virus neutralization—mechanism, stoichiometry, kinetics, and efficacy—from surprising new angles.

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Neutralization Properties of Simian Immunodeficiency Viruses Infecting Chimpanzees and Gorillas

mBio ◽

10.1128/mbio.00296-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 17

Author(s):

Hannah J. Barbian ◽

Julie M. Decker ◽

Frederic Bibollet-Ruche ◽

Rachel P. Galimidi ◽

Anthony P. West ◽

...

Keyword(s):

T Cells ◽

Binding Site ◽

Pan Troglodytes ◽

Neutralizing Antibodies ◽

Gorilla Gorilla ◽

Protective Capacity ◽

Content Type ◽

Ccr5 Receptor ◽

Cd4 Binding Site ◽

Hiv 1

ABSTRACTBroadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytestroglodytes) (SIVcpzPtt) and eastern (Pan troglodytesschweinfurthii) (SIVcpzPts) chimpanzees (n= 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n= 1). We found that bNabs directed against the CD4 binding site (n= 10), peptidoglycans at the base of variable loop 3 (V3) (n= 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n= 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n= 3) as well as llama-derived (heavy chain only) antibodies (n= 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPttstrains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Igmim2, CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4+T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection.IMPORTANCESIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4+T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.

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Broad neutralization by a combination of antibodies recognizing the CD4 binding site and a new conformational epitope on the HIV-1 envelope protein

Journal of Experimental Medicine ◽

10.1084/jem.20120423 ◽

2012 ◽

Vol 209 (8) ◽

pp. 1469-1479 ◽

Cited By ~ 123

Author(s):

Florian Klein ◽

Christian Gaebler ◽

Hugo Mouquet ◽

D. Noah Sather ◽

Clara Lehmann ◽

...

Keyword(s):

Binding Site ◽

Envelope Protein ◽

Neutralizing Antibodies ◽

Conformational Epitope ◽

Broadly Neutralizing Antibodies ◽

Cd4 Binding Site ◽

Capture Method ◽

Anti Hiv ◽

Hiv 1 ◽

Viral Isolates

Two to three years after infection, a fraction of HIV-1–infected individuals develop serologic activity that neutralizes most viral isolates. Broadly neutralizing antibodies that recognize the HIV-1 envelope protein have been isolated from these patients by single-cell sorting and by neutralization screens. Here, we report a new method for anti–HIV-1 antibody isolation based on capturing single B cells that recognize the HIV-1 envelope protein expressed on the surface of transfected cells. Although far less efficient than soluble protein baits, the cell-based capture method identified antibodies that bind to a new broadly neutralizing epitope in the vicinity of the V3 loop and the CD4-induced site (CD4i). The new epitope is expressed on the cell surface form of the HIV-1 spike, but not on soluble forms of the same envelope protein. Moreover, the new antibodies complement the neutralization spectrum of potent broadly neutralizing anti-CD4 binding site (CD4bs) antibodies obtained from the same individual. Thus, combinations of potent broadly neutralizing antibodies with complementary activity can account for the breadth and potency of naturally arising anti–HIV-1 serologic activity. Therefore, vaccines aimed at eliciting anti–HIV-1 serologic breadth and potency should not be limited to single epitopes.

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