Influence of Novel CD4 Binding-Defective HIV-1 Envelope Glycoprotein Immunogens on Neutralizing Antibody and T-Cell Responses in Nonhuman Primates

ABSTRACT The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.

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Characterization and Implementation of a Diverse Simian Immunodeficiency Virus SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multimodal Vaccines Expressing the SIVmac239 Envelope

Journal of Virology ◽

10.1128/jvi.01221-14 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8130-8151 ◽

Cited By ~ 18

Author(s):

Katie M. Kilgore ◽

Megan K. Murphy ◽

Samantha L. Burton ◽

Katherine S. Wetzel ◽

S. Abigail Smith ◽

...

Keyword(s):

Simian Immunodeficiency Virus ◽

Nonhuman Primate ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Neutralizing Antibody ◽

Cd40 Ligand ◽

Antibody Activity ◽

Immunodeficiency Virus ◽

Antibody Profile ◽

Hiv 1

ABSTRACTAntibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen.IMPORTANCEMany in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.

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Elicitation of Cluster A and Co-Receptor Binding Site Antibodies Are Required to Eliminate HIV-1 Infected Cells

Microorganisms ◽

10.3390/microorganisms8050710 ◽

2020 ◽

Vol 8 (5) ◽

pp. 710 ◽

Cited By ~ 2

Author(s):

Guillaume Beaudoin-Bussières ◽

Jérémie Prévost ◽

Gabrielle Gendron-Lepage ◽

Bruno Melillo ◽

Junhua Chen ◽

...

Keyword(s):

Binding Site ◽

Envelope Glycoprotein ◽

Antibody Dependent Cellular Cytotoxicity ◽

Receptor Binding Site ◽

Coreceptor Binding ◽

Infected Cells ◽

Cluster A ◽

Hiv 1 ◽

Coreceptor Binding Site

HIV-1-infected individuals raise a polyclonal antibody response targeting multiple envelope glycoprotein (Env) epitopes. Interestingly, two classes of non-neutralizing CD4-induced (CD4i) antibodies, present in the majority of HIV-1-infected individuals have been described to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of small CD4 mimetic compounds (CD4mc). These antibodies recognize the coreceptor binding site (CoRBS) and the constant region one and two (C1C2 or inner domain cluster A) of the gp120. In combination with CD4mc they have been shown to stabilize an antibody-vulnerable Env conformation, known as State 2A. Here we evaluated the importance of these two families of Abs in ADCC responses by immunizing guinea pigs with gp120 immunogens that have been modified to elicit or not these types of antibodies. Underlying the importance of anti-CoRBS and anti-cluster A Abs in stabilizing State 2A, ADCC responses were only observed in the presence of these two types of CD4i antibodies. Altogether, our results suggest that these two families of CD4i antibodies must be taken into account when considering future strategies relying on the use of CD4mc to eliminate HIV-1-infected cells in vivo.

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Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

Journal of Virology ◽

10.1128/jvi.02428-16 ◽

2017 ◽

Vol 91 (9) ◽

Cited By ~ 39

Author(s):

Eden P. Go ◽

Haitao Ding ◽

Shijian Zhang ◽

Rajesh P. Ringe ◽

Nathan Nicely ◽

...

Keyword(s):

Cho Cells ◽

Envelope Glycoprotein ◽

Neutralizing Antibody ◽

Nitrilotriacetic Acid ◽

Viral Envelope ◽

Purification Method ◽

Glycosylation Profile ◽

Glycosylation Sites ◽

The Impact ◽

Hiv 1

ABSTRACT HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells. IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the “target” glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a “glycosylation target” for their immunogens, and they show how protein production variables can impact Env glycosylation.

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The Envelope Glycoprotein Ectodomains Determine the Efficiency of CD4+ T Lymphocyte Depletion in Simian– Human Immunodeficiency Virus–Infected Macaques

Journal of Experimental Medicine ◽

10.1084/jem.188.6.1159 ◽

1998 ◽

Vol 188 (6) ◽

pp. 1159-1171 ◽

Cited By ~ 89

Author(s):

Gunilla B. Karlsson ◽

Matilda Halloran ◽

Dominik Schenten ◽

Juliette Lee ◽

Paul Racz ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Lymphocytes ◽

T Lymphocyte ◽

Envelope Glycoprotein ◽

Rhesus Macaques ◽

Immune Deficiency Syndrome ◽

Lymphocyte Depletion ◽

Immunodeficiency Virus ◽

Hiv 1

CD4+ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)–infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian–human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4+ T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4+ T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4+ T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.

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Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 Envelope glycoprotein trimers on neutralizing antibody induction

Virology ◽

10.1016/j.virol.2017.02.024 ◽

2017 ◽

Vol 505 ◽

pp. 193-209 ◽

Cited By ~ 16

Author(s):

Ema T. Crooks ◽

Keiko Osawa ◽

Tommy Tong ◽

Samantha L. Grimley ◽

Yang D. Dai ◽

...

Keyword(s):

Binding Site ◽

Envelope Glycoprotein ◽

Neutralizing Antibody ◽

Cd4 Binding Site ◽

Antibody Induction ◽

Hiv 1

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Immunization with Wild-Type or CD4-Binding-Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus

Journal of Virology ◽

10.1128/jvi.01015-10 ◽

2010 ◽

Vol 84 (18) ◽

pp. 9086-9095 ◽

Cited By ~ 13

Author(s):

Christopher Sundling ◽

Sijy O'Dell ◽

Iyadh Douagi ◽

Mattias N. Forsell ◽

Andreas Mörner ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Specific Binding ◽

Rhesus Macaques ◽

Direct Interaction ◽

Challenge Virus ◽

Heterologous Challenge ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model.

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Polyclonal Broadly Neutralizing Antibody Activity Characterized by CD4 Binding Site and V3-Glycan Antibodies in a Subset of HIV-1 Virus Controllers

Frontiers in Immunology ◽

10.3389/fimmu.2021.670561 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tinashe E. Nyanhete ◽

Robert J. Edwards ◽

Celia C. LaBranche ◽

Katayoun Mansouri ◽

Amanda Eaton ◽

...

Keyword(s):

Binding Site ◽

Neutralizing Antibodies ◽

Humoral Response ◽

Neutralizing Antibody ◽

Small Subset ◽

Antibody Activity ◽

Viral Loads ◽

Immune Pressure ◽

Cd4 Binding Site ◽

Hiv 1

Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication [Virus Controllers (VCs)]. Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of envs from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection.

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In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

Virology ◽

10.1016/j.virol.2007.10.044 ◽

2008 ◽

Vol 372 (2) ◽

pp. 409-420 ◽

Cited By ~ 26

Author(s):

Maria Luisa Visciano ◽

Michael Tuen ◽

Miroslaw K. Gorny ◽

Catarina E. Hioe

Keyword(s):

Binding Site ◽

Envelope Glycoprotein ◽

Envelope Glycoprotein Gp120 ◽

Cd4 Binding Site ◽

Glycoprotein Gp120 ◽

Humoral Responses ◽

Hiv 1

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A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo

PLoS Pathogens ◽

10.1371/journal.ppat.1005238 ◽

2015 ◽

Vol 11 (10) ◽

pp. e1005238 ◽

Cited By ~ 29

Author(s):

Natalia T. Freund ◽

Joshua A. Horwitz ◽

Lilian Nogueira ◽

Stuart A. Sievers ◽

Louise Scharf ◽

...

Keyword(s):

Binding Site ◽

Neutralizing Antibody ◽

Cd4 Binding Site ◽

Hiv 1

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Peptides presenting the binding site of human CD4 for the HIV-1 envelope glycoprotein gp120

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.8.214 ◽

2012 ◽

Vol 8 ◽

pp. 1858-1866 ◽

Cited By ~ 4

Author(s):

Julia Meier ◽

Kristin Kassler ◽

Heinrich Sticht ◽

Jutta Eichler

Keyword(s):

Amino Acids ◽

Binding Site ◽

Envelope Glycoprotein ◽

Conformational Stability ◽

Cellular Receptor ◽

Binding Assays ◽

Glycoprotein Gp120 ◽

Dynamics Simulations ◽

Key Residues ◽

Hiv 1

Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide–gp120 complexes. The specificity of the peptide–gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids.

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