scholarly journals Quantifying Anti-HIV Envelope-Specific Antibodies in Plasma from HIV Infected Individuals

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 487 ◽  
Author(s):  
Sanket Kant ◽  
Ningyu Zhang ◽  
Jean-Pierre Routy ◽  
Cécile Tremblay ◽  
Réjean Thomas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Elite Controllers ◽  
Specific Antibodies ◽  
Viral Loads ◽  
Monomeric Gp120 ◽  
Infected Cells ◽  
Bystander Cells ◽  
Hiv Envelope ◽  
Anti Hiv

Quantifying HIV Envelope (Env)-specific antibodies in HIV+ plasma is useful for interpreting antibody dependent cellular cytotoxicity assay results. HIV Env, the only viral protein expressed on the surface of infected cells, has a native trimeric closed conformation on cells infected with wild-type HIV. However, CD4+ uninfected bystander cells in HIV+ cell cultures bind gp120 shed from HIV+ cells exposing CD4-induced epitopes normally hidden in native Env. We used flow-cytometry based assays to quantify antibodies in HIV+ plasma specific for native trimeric Env or gp120/CD4 conjugates using CEM.NKr.CCR5 (CEM) cells infected with HIV (iCEM) or coated with recombinant gp120 (cCEM), as a surrogate for gp120+ HIV- bystander cells. Results from both assays were compared to those of a plate-based ELISA to monomeric gp120. The levels of Env-specific antibodies to cCEM and iCEM, measured by flow cytometry, and to gp120 by ELISA were positively correlated. More antibodies in HIV+ plasma recognized the gp120 conformation exposed on cCEM than on iCEM. Comparisons of plasma from untreated progressors, treated progressors, and elite controllers revealed that antibodies to Env epitopes were the lowest in treated progressors. Plasma from elite controllers and untreated progressors had similarly high levels of Env-specific antibodies, despite elite controllers having undetectable HIV viral loads, while untreated progressors maintained high viral loads.

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.

Evolution of antibodies to native trimeric envelope and their Fc dependent functions in untreated and treated primary HIV infection

2021 ◽  
Author(s):  
Lauren Nagel ◽  
Sanket Kant ◽  
Christopher Leeks ◽  
Jean-Pierre Routy ◽  
Cécile Tremblay ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Specific Antibody ◽  
People Living With Hiv ◽  
Specific Antibodies ◽  
Closed Conformation ◽  
Art Initiation ◽  
Infected Cells ◽  
Bystander Cells ◽  
Living With Hiv ◽  
Antibody Levels

People living with HIV (PLWH) develop both anti-Envelope-specific antibodies, which bind the closed trimeric HIV Envelope present on infected cells and anti-gp120-specific antibodies, which bind gp120 monomers shed by infected cells and taken up by CD4 on uninfected bystander cells. Both antibodies have an Fc portion that binds to Fc Receptors on several types of innate immune cells and stimulates them to develop anti-viral functions. Among these Fc dependent functions (FcDFs) are antibody dependent (AD) cellular cytotoxicity (ADCC), AD cellular trogocytosis (ADCT) and AD phagocytosis (ADCP). Here, we assessed the evolution of total immunoglobulin G (IgG), anti-gp120 and anti-Envelope IgG antibodies and their FcDFs in plasma samples from anti-retroviral therapy (ART) naïve subjects during early HIV infection (28-194 days post infection [DPI]). We found that both the concentrations and FcDFs of anti-gp120 and anti-Envelope antibodies increased with time in ART-naïve PLWH. Although generated concurrently, anti-gp120-specific antibodies were 20.7-fold more abundant than anti-Envelpe-specific antibodies, both specificities being strongly correlated with each other and FcDFs. Among the FcDFs, only ADCP activity was inversely correlated with concurrent viral load. PLWH who started ART >90 DPI showed higher anti-Envelope-specific antibody levels, ADCT and ADCP activities than those starting ART <90 DPI. However, in longitudinally collected samples, ART initiation at >90 DPI was accompanied by a faster decline in anti-Envelope-specific antibody levels, which did not translate to a faster decline in FcDFs compared to those starting ART <90 DPI. IMPORTANCE Closed conformation Envelope is expressed on the surface of HIV-infected cells. Antibodies targeting this conformation and that support FcDFs have the potential to control HIV. This study tracks the timing of the appearance and evolution of antibodies to closed conformation Envelope, whose concentration increases over the first 6 mos of infection. Antiretroviral therapy (ART) initiation blunts further increases in the concentration of these antibodies and their and FcDFs. However, antibodies to open conformation Envelope also increase with DPI until ART initiation. These antibodies target uninfected bystander cells, which may contribute to loss of uninfected CD4 cells and pathogenicity. This manuscript presents, for the first time, the evolution of antibodies to closed conformation Envelope and their fate on-ART. This information may be useful in making decisions on the timing of ART initiation in early HIV infection.

scholarly journals Efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective action of complement factor H and decay accelerating factor (DAF, CD55).

1996 ◽  
Vol 183 (1) ◽  
pp. 307-310 ◽  
Author(s):  
H Stoiber ◽  
C Pintér ◽  
A G Siccardi ◽  
A Clivio ◽  
M P Dierich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Serum ◽  
Factor H ◽  
Complement Factor H ◽  
Complement Factor ◽  
Human Complement ◽  
Specific Antibodies ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv Envelope

Activation of the human complement system leads to complement deposition on human immunodeficiency virus (HIV) and HIV-infected cells without causing efficient complement-mediated lysis. Even in the presence of HIV-specific antibodies, only a few particles are destroyed, demonstrating that HIV is intrinsically resistant to human complement. Here we report that, in addition to decay accelerating factor (DAF) being partially responsible, human complement factor H (CFH), a humoral negative regulator of complement activation, is most critical for this resistance. In the presence of HIV-specific antibodies, sera devoid of CFH (total genetic deficiency or normal human serum depleted of CFH by affinity chromatography) lysed free virus and HIV-infected but not uninfected cells. In the presence of CFH, lysis of HIV was only obtained when binding of CFH to gp41 was inhibited by a monoclonal antibody against a main CFH-binding site in gp41. Since CFH is an abundant protein in serum, and high local concentration of CFH can be obtained at the surface of HIV as the result of specific interactions of CFH with the HIV envelope, it is proposed that the resistance of HIV and HIV-infected cells against complement-mediated lysis in vivo is dependent on DAF and CFH and can be overcome by suppressing this protection. Neutralization of HIV may be achieved by antibodies against DAF and, more importantly, antibodies against CFH-binding sites on HIV envelope proteins.

scholarly journals The HIV-1 gp120 CD4-Bound Conformation Is Preferentially Targeted by Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Sera from HIV-1-Infected Individuals

2014 ◽  
Vol 89 (1) ◽  
pp. 545-551 ◽  
Author(s):  
Maxime Veillette ◽  
Mathieu Coutu ◽  
Jonathan Richard ◽  
Laurie-Anne Batraville ◽  
Olina Dagher ◽  
...  
Keyword(s):  
Vaccine Trial ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Variable Regions ◽  
Effector Functions ◽  
High Prevalence ◽  
Infected Cells ◽  
Hiv Envelope ◽  
Hiv 1

ABSTRACTRecent studies have linked antibody Fc-mediated effector functions with protection or control of human immunodeficiency type 1 (HIV-1) and simian immunodeficiency (SIV) infections. Interestingly, the presence of antibodies with potent antibody-dependent cellular cytotoxicity (ADCC) activity in the Thai RV144 vaccine trial was suggested to correlate with decreased HIV-1 acquisition risk. These antibodies recently were found to recognize HIV envelope (Env) epitopes exposed upon Env-CD4 interaction. CD4 downregulation by Nef and Vpu, as well as Vpu-mediated BST-2 antagonism, were reported to modulate exposure of those CD4-induced HIV-1 Env epitopes and were proposed to play a role in reducing the susceptibility of infected cells to ADCC mediated by this class of antibodies. Here, we report the high prevalence of antibodies recognizing CD4-induced HIV-1 Env epitopes in sera from HIV-1-infected individuals, which correlated with their ability to mediate ADCC responses against HIV-1-infected cells, exposing these Env epitopes at the cell surface. Furthermore, our results indicate that Env variable regions V1, V2, V3, and V5 do not represent a major determinant for ADCC responses mediated by sera from HIV-1-infected individuals. Altogether, these findings suggest that HIV-1 tightly controls the exposure of certain Env epitopes at the surface of infected cells in order to prevent elimination by Fc-effector functions.IMPORTANCEHere, we identified a particular conformation of HIV-1 Env that is specifically targeted by ADCC-mediating antibodies present in sera from HIV-1-infected individuals. This observation suggests that HIV-1 developed sophisticated mechanisms to minimize the exposure of these epitopes at the surface of infected cells.

scholarly journals Detection of the HIV-1 accessory proteins Nef and Vpu by flow cytometry represents a new tool to study their functional interplay within a single infected CD4+ T cell

2021 ◽  
Author(s):  
Jeremie Prevost ◽  
Jonathan Richard ◽  
Romain Gasser ◽  
Halima Medjahed ◽  
Frank Kirchhoff ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd4 T Cells ◽  
Staining Technique ◽  
Accessory Proteins ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Infected Cells ◽  
Intracellular Detection ◽  
Infectious Molecular Clones ◽  
Hiv 1

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells.

scholarly journals Lack of ADCC Breadth of Human Nonneutralizing Anti-HIV-1 Antibodies

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Timothée Bruel ◽  
Florence Guivel-Benhassine ◽  
Valérie Lorin ◽  
Hugues Lortat-Jacob ◽  
Françoise Baleux ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Hiv Positive ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Broadly Neutralizing Antibodies ◽  
Efficacy Trial ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Rv144 Vaccine

ABSTRACT Anti-human immunodeficiency virus type 1 (HIV-1) nonneutralizing antibodies (nnAbs) capable of antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. Broadly neutralizing antibodies (bNAbs) also mediate ADCC in cell culture and rely on their Fc region for optimal efficacy in animal models. Here, we selected 9 monoclonal nnAbs and 5 potent bNAbs targeting various epitopes and conformations of the gp120/41 complex and analyzed the potency of the two types of antibodies to bind and eliminate HIV-1-infected cells in culture. Regardless of their neutralizing activity, most of the selected antibodies recognized and killed cells infected with two laboratory-adapted HIV-1 strains. Some nnAbs also bound bystander cells that may have captured viral proteins. However, in contrast to the bNAbs, the nnAbs bound poorly to reactivated infected cells from 8 HIV-positive individuals and did not mediate effective ADCC against these cells. The nnAbs also inefficiently recognize cells infected with 8 different transmitted-founder (T/F) isolates. The addition of a synthetic CD4 mimetic enhanced the binding and killing efficacy of some of the nnAbs in an epitope-dependent manner without reaching the levels achieved by the most potent bNAbs. Overall, our data reveal important qualitative and quantitative differences between nnAbs and bNAbs in their ADCC capacity and strongly suggest that the breadth of recognition of HIV-1 by nnAbs is narrow. IMPORTANCE Most of the anti-HIV antibodies generated by infected individuals do not display potent neutralizing activities. These nonneutralizing antibodies (nnAbs) with antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. However, in primate models, the nnAbs do not protect against simian-human immunodeficiency virus (SHIV) acquisition. Thus, the role of nnAbs with ADCC activity in protection from infection remains debatable. In contrast, broadly neutralizing antibodies (bNAbs) neutralize a large array of viral strains and mediate ADCC in cell culture. We analyzed the capacities of 9 nnAbs and 5 bNAbs to eliminate infected cells. We selected 18 HIV-1 strains, including virus reactivated from the reservoir of HIV-positive individuals and transmitted-founder isolates. We report that the nnAbs bind poorly to cells infected with primary HIV-1 strains and do not mediate potent ADCC. Overall, our data show that the breadth of recognition of HIV-1 by nnAbs is narrow.

scholarly journals Antibody-Dependent Cellular Cytotoxicity-Competent Antibodies against HIV-1-Infected Cells in Plasma from HIV-Infected Subjects

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Franck P. Dupuy ◽  
Sanket Kant ◽  
Alexandre Barbé ◽  
Jean-Pierre Routy ◽  
Julie Bruneau ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Cell Types ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Adcc Assay ◽  
Cd4 Binding Site ◽  
Infected Cells ◽  
Bystander Cells ◽  
Hiv 1

ABSTRACT Measuring Envelope (Env)-specific antibody (Ab)-dependent cellular cytotoxicity (ADCC)-competent Abs in HIV+ plasma is challenging because Env displays distinctive epitopes when present in a native closed trimeric conformation on infected cells or in a CD4-bound conformation on uninfected bystander cells. We developed an ADCC model which distinguishes Env-specific ADCC-competent Abs based on their capacity to eliminate infected, bystander, or Env rgp120-coated cells as a surrogate for shed gp120 on bystander cells. A panel of monoclonal Abs (MAbs), used to opsonize these target cells, showed that infected cells were preferentially recognized/eliminated by MAbs to CD4 binding site, V3 loop, and viral spike epitopes whereas bystander/coated cells were preferentially recognized/eliminated by Abs to CD4-induced (CD4i) epitopes. In HIV-positive (HIV+) plasma, Env-specific Abs recognized and supported ADCC of infected cells, though a majority were directed toward CD4i epitopes on bystander cells. For ADCC activity to be effective in HIV control, ADCC-competent Abs need to target genuinely infected cells. IMPORTANCE HIV Env-specific nonneutralizing Abs (NnAbs) able to mediate ADCC have been implicated in protection from HIV infection. However, Env-specific NnAbs have the capacity to support ADCC of both HIV-infected and HIV-uninfected bystander cells, potentially leading to misinterpretations when the assay used to measure ADCC does not distinguish between the two target cell types present in HIV cultures. Using a novel ADCC assay, which simultaneously quantifies the killing activity of Env-specific Abs on both infected and uninfected bystander cells, we observed that only a minority of Env-specific Abs in HIV+ plasma mediated ADCC of genuinely HIV-infected cells displaying Env in its native closed conformation. This assay can be used for the development of vaccine strategies aimed at eliciting Env-specific Ab responses capable of controlling HIV infection.

scholarly journals Nef Proteins from HIV-1 Elite Controllers Are Inefficient at Preventing Antibody-Dependent Cellular Cytotoxicity

2015 ◽  
Vol 90 (6) ◽  
pp. 2993-3002 ◽  
Author(s):  
Nirmin Alsahafi ◽  
Shilei Ding ◽  
Jonathan Richard ◽  
Tristan Markle ◽  
Nathalie Brassard ◽  
...  
Keyword(s):  
Viral Replication ◽  
Antiretroviral Treatment ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Elite Controllers ◽  
Viremia Level ◽  
Infected Cells ◽  
Hiv 1 ◽  
Durable Suppression ◽  
Increased Susceptibility

ABSTRACTImpairment of Nef function, including reduced CD4 downregulation, was described in a subset of HIV-1-infected individuals that control viral replication without antiretroviral treatment (elite controllers [EC]). Elimination of HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) requires the presence of envelope glycoproteins (Env) in the CD4-bound conformation, raising the possibility that accumulating CD4 at the surface of virus-infected cells in EC could interact with Env and thereby sensitize these cells to ADCC. We observed a significant increase in the exposure of Env epitopes targeted by ADCC-mediating antibodies at the surface of cells expressing Nef isolates from EC; this correlated with enhanced susceptibility to ADCC. Altogether, our results suggest that enhanced susceptibility of HIV-1-infected cells to ADCC may contribute to the EC phenotype.IMPORTANCENef clones derived from elite controllers (EC) have been shown to be attenuated for CD4 downregulation; how this contributes to the nonprogressor phenotype of these infected individuals remains uncertain. Increasing evidence supports a role for HIV-specific antibody-dependent cellular cytotoxicity (ADCC) in controlling viral infection and replication. Here, we show that residual CD4 left at the surface of cells expressing Nef proteins isolated from ECs are sufficient to allow Env-CD4 interaction, leading to increased exposure of Env CD4-induced epitopes and increased susceptibility of infected cells to ADCC. Our results suggest that ADCC might be an active immune mechanism in EC that helps to maintain durable suppression of viral replication and low plasma viremia level in this rare subset of infected individuals. Therefore, targeting Nef's ability to downregulate CD4 could render HIV-1-infected cells susceptible to ADCC and thus have therapeutic utility.

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