scholarly journals Across functional boundaries: making non-neutralizing antibodies to neutralize HIV-1 and mediate Fc-mediated effector killing of infected cells

2021 ◽  
Author(s):  
Jonathan Richard ◽  
Dung Nguyen ◽  
William D. Tolbert ◽  
Romain Gasser ◽  
shilei ding ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Biological Activities ◽  
Single Chain ◽  
Receptor Binding Site ◽  
Antibody Recognition ◽  
Tier 1 ◽  
Competition Binding ◽  
Infected Cells ◽  
Cluster A ◽  
Hiv 1

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or Cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, closed Env trimer present on HIV-1 virions or expressed on HIV-1 infected cells. Antibodies targeting this region are therefore non-neutralizing and their potential as mediators of antibody depended cellular cytoxicity (ADCC) of HIV-1 infected cells diminished by a lack of available binding targets. Here we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or Cluster A specificity to the extracellular domain 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening closed Env trimers on HIV-1 particles/infected cells to expose the Cluster A region and activate ADCC and neutralization against these non-neutralizing targets.

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

2020 ◽  
Vol 8 (5) ◽  
pp. 710 ◽  
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.

scholarly journals eCD4-Ig Variants That More Potently Neutralize HIV-1

2018 ◽  
Vol 92 (12) ◽  
Author(s):  
Ina Fetzer ◽  
Matthew R. Gardner ◽  
Meredith E. Davis-Gardner ◽  
Neha R. Prasad ◽  
Barnett Alfant ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Entry Inhibitor ◽  
Immune Effector ◽  
Effector Cells ◽  
Antibody Recognition ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Prophylaxis Therapy ◽  
Hiv 1

ABSTRACTThe human immunodeficiency virus type 1 (HIV-1) entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralized all HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates that it has been tested against, suggesting that it may be useful in clinical settings, where antibody escape is a concern. Here, we characterize three new eCD4-Ig variants, each with a different architecture and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as the original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented the promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications.IMPORTANCEHIV-1 bNAbs have properties different from those of antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral life cycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the utility of antibodies as a treatment for HIV-1 infection or as part of an effort to eradicate latently infected cells. eCD4-Ig is an antibody-like entry inhibitor that closely mimics HIV-1's obligate receptors. eCD4-Ig appears to be qualitatively different from antibodies, since it neutralizes all HIV-1, HIV-2, and SIV isolates. Here, we characterize three new structurally distinct eCD4-Ig variants and show that each excels in a key property useful to prevent, treat, or cure an HIV-1 infection. For example, one variant neutralized HIV-1 most efficiently, while others best enlisted natural killer cells to eliminate infected cells. These observations will help generate eCD4-Ig variants optimized for different clinical applications.

scholarly journals Uninfected Bystander Cells Impact the Measurement of HIV-Specific Antibody-Dependent Cellular Cytotoxicity Responses

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Jonathan Richard ◽  
Jérémie Prévost ◽  
Amy E. Baxter ◽  
Benjamin von Bredow ◽  
Shilei Ding ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Antibody Recognition ◽  
Vaccine Trials ◽  
Infected Cells ◽  
Bystander Cells ◽  
Hiv 1

ABSTRACTThe conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a “closed” conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affectin vitromeasurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells inin vitrocultures orex vivosamples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands.IMPORTANCEEmerging evidence supports a role for antibody-dependent cellular cytotoxicity (ADCC) in protection against HIV-1 transmission and disease progression. However, there are conflicting reports regarding the ability of nonneutralizing antibodies targeting CD4-inducible (CD4i) Env epitopes to mediate ADCC. Here, we performed a side-by-side comparison of different methods currently being used in the field to measure ADCC responses to HIV-1. We found that assays which are unable to differentiate virus-infected from uninfected cells greatly overestimate ADCC responses mediated by antibodies to CD4i epitopes and underestimate responses mediated by broadly neutralizing antibodies (bNAbs). Our results strongly argue for the use of assays that measure ADCC against HIV-1-infected cells expressing physiologically relevant conformations of Env to evaluate correlates of protection in vaccine trials.

scholarly journals Stabilizing the HIV-1 envelope glycoprotein State 2A conformation

2020 ◽  
Author(s):  
Dani Vézina ◽  
Shang Yu Gong ◽  
William D. Tolbert ◽  
Shilei Ding ◽  
Dung Nguyen ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Neutralizing Antibodies ◽  
Coreceptor Binding ◽  
Viral Particles ◽  
A Cell ◽  
Infected Cells ◽  
Cluster A ◽  
State 1 ◽  
Hiv 1 ◽  
Conformation State

The HIV-1 envelope glycoprotein (Env) trimer [(gp120/gp41)3] is a metastable complex expressed at the surface of viral particles and infected cells that samples different conformations. Before engaging CD4, Env adopts an antibody-resistant “closed” conformation (State 1). CD4 binding triggers an intermediate conformation (State 2) and then a more “open” conformation (State 3) that can be recognized by non-neutralizing antibodies (nnAbs) such as those that recognize the coreceptor binding site (CoRBS). Binding of antibodies to the CoRBS permits another family of nnAbs, the anti-cluster A family of Abs which target the gp120 inner domain, to bind and stabilize an asymmetric conformation (State 2A). Cells expressing Env in this conformation are susceptible to antibody-dependent cellular cytotoxicity (ADCC). This conformation can be stabilized by small-molecule CD4 mimetics (CD4mc) or soluble CD4 (sCD4) in combination with anti-CoRBS Ab and anti-cluster A antibodies. The precise stoichiometry of each component that permits this sequential opening of Env remains unknown. Here, we used a cell-based ELISA (CBE) assay to evaluate each component individually. In this assay we used a “trimer mixing” approach by combining wild-type (wt) subunits with subunits impaired for CD4 or CoRBS Ab binding. This enabled us to show that State 2A requires all three gp120 subunits to be bound by sCD4/CD4mc and anti-CoRBS Abs. Two of these subunits can then bind anti-cluster A Abs. Altogether, our data suggests how this antibody vulnerable Env conformation is stabilized. Importance Stabilization of HIV-1 Env State 2A has been shown to sensitize infected cells to ADCC. State 2A can be stabilized by a “cocktail” composed of CD4mc, anti-CoRBS and anti-cluster A Abs. We present evidence that optimal State 2A stabilization requires all three gp120 subunits to be bound by both CD4mc and anti-CoRBS Abs. Our study provides valuable information on how to stabilize this ADCC-vulnerable conformation. Strategies aimed at stabilizing State 2A might have therapeutic utility.

scholarly journals Across Functional Boundaries: Making Nonneutralizing Antibodies To Neutralize HIV-1 and Mediate Fc-Mediated Effector Killing of Infected Cells

mBio ◽  
2021 ◽  
Author(s):  
Jonathan Richard ◽  
Dung N. Nguyen ◽  
William D. Tolbert ◽  
Romain Gasser ◽  
Shilei Ding ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Host Cell ◽  
Constant Region ◽  
Antibody Recognition ◽  
Coreceptor Binding ◽  
Infected Cells ◽  
Cluster A ◽  
Hiv 1 ◽  
Coreceptor Binding Site

Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity.

Functional analysis of a monoclonal antibody reactive against anti-cluster A epitope obtained from a patient infected with HIV-1 CRF02_AG

2020 ◽  
Author(s):  
Hasan Md Za ◽  
Takeo Kuwata ◽  
Shokichi Takahama ◽  
Kaku Yu ◽  
Shaswata Biswas ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Infected Individual ◽  
Adcc Activity ◽  
Subtype B ◽  
Coreceptor Binding ◽  
Infected Cells ◽  
Cluster A ◽  
Hiv 1

Abstract Background: Recent data suggest the importance of non-neutralizing antibodies (nnAbs) in the development of vaccines against HIV-1 because two types of nnAbs that recognize the coreceptor binding site (CoRBS) and inner domain cluster A mediate antibody-dependent cellular-cytotoxicity (ADCC) against HIV-1-infected cells. However, many studies have been conducted with nnAbs obtained from subtype B-infected individuals, with few studies in patients with non-subtype B infections. Results: We isolated a monoclonal antibody 1E5 from a CRF02_AG-infected individual and constructed two forms of antibody with constant regions of IgG1 or IgG3. The epitope of 1E5 belongs to cluster A, which consists of C1 and C2 of gp120, and 1E5 binds to 27 out of 35 strains (77%) across the subtypes. The 1E5 showed strong ADCC activity, especially in the form of IgG3 in the presence of small CD4-mimetic compounds (CD4mc) and anti-CoRBS antibody, but did not show any neutralizing activity even against the isolates with strong binding activities. The enhancement in the binding of A32, anti-cluster A antibody isolated from a patient with subtype B infection, was observed in the presence of 1E5 and the combination of both anti-cluster A antibodies enhanced ADCC activity. Conclusions: These results suggest that anti-cluster A antibodies that are induced in patients with different HIV-1 subtype infections have common functional modality and may have unexpected interactions. These data may have implications for vaccine development against HIV-1.

scholarly journals Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo

Science ◽  
2016 ◽  
Vol 352 (6288) ◽  
pp. 1001-1004 ◽  
Author(s):  
C.-L. Lu ◽  
D. K. Murakowski ◽  
S. Bournazos ◽  
T. Schoofs ◽  
D. Sarkar ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Broadly Neutralizing Antibodies ◽  
Infected Cells ◽  
Hiv 1

scholarly journals Antibody-Induced Internalization of HIV-1 Env Proteins Limits Surface Expression of the Closed Conformation of Env

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Sai Priya Anand ◽  
Jonathan R. Grover ◽  
William D. Tolbert ◽  
Jérémie Prévost ◽  
Jonathan Richard ◽  
...  
Keyword(s):  
Immune Responses ◽  
Envelope Glycoprotein ◽  
Neutralizing Antibodies ◽  
Surface Expression ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Broadly Neutralizing Antibodies ◽  
Closed Conformation ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here, we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of antibody-dependent cellular cytotoxicity (ADCC). Using flow cytometry and confocal microscopy, we show that broadly neutralizing antibodies (bNAbs) targeting the “closed” conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibilities of infected cells to ADCC. Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the “closed” conformation of Env expressed on HIV-1-infected cells. IMPORTANCE HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus, a better understanding of this mechanism might help develop antibodies with improved capacities to mediate ADCC.

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