Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.     

HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization

Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01.     

Neutralizing Monoclonal Antibodies Block Human Immunodeficiency Virus Type 1 Infection of Dendritic Cells and Transmission to T Cells

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14⁺ blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3⁺ T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.     

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4⁺ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

Antibody to gp41 MPER Alters Functional Properties of HIV-1 Env without Complete Neutralization

Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.     

HIV-1 Autologous Antibody Neutralization Associates with Mother to Child Transmission

The HIV-1 characteristics associated with mother to child transmission (MTCT) are still poorly understood and if known would indicate where intervention strategies should be targeted. In contrast to horizontally infected individuals, exposed infants possess inherited antibodies (Abs) from their mother with the potential to protect against infection. We investigated the HIV-1 gp160 envelope proteins from seven transmitting mothers (TM) whose children were infected either during gestation or soon after delivery and from four non-transmitting mothers (NTM) with similar viral loads and CD4 counts. Using pseudo-typed viruses we tested gp160 envelope glycoproteins for TZM-bl infectivity, CD4 and CCR5 interactions, DC-SIGN capture and transfer and neutralization with an array of common neutralizing Abs (NAbs) (2F5, 2G12, 4E10 and b12) as well as mother and infant plasma. We found no viral correlates associated with HIV-1 MTCT nor did we find differences in neutralization with the panel of NAbs. We did, however, find that TM possessed significantly higher plasma neutralization capacities than NTM (P  = 0.002). Furthermore, we found that in utero (IU) TM had a higher neutralization capacity than mothers transmitting either peri - partum (PP) or via breastfeeding (BF) (P  = 0.002). Plasma from children infected IU neutralized viruses carrying autologous gp160 viral envelopes as well as those from their corresponding mothers whilst plasma from children infected PP and/or BF demonstrated poor neutralizing capacity. Our results demonstrate heightened autologous NAb responses against gp120/gp41 can associate with a greater risk of HIV-1 MTCT and more specifically in those infants infected IU. Although the number of HIV-1 transmitting pairs is low our results indicate that autologous NAb responses in mothers and infants do not protect against MTCT and may in fact be detrimental when considering IU HIV-1 transmissions.     

HIV-1 Capture and Transmission by Dendritic Cells: The Role of Viral Glycolipids and the Cellular Receptor Siglec-1

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.     

Mechanism of HIV-1 Neutralization by Antibodies Targeting a Membrane-Proximal Region of gp41

Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.     

Broadly Neutralizing Antibody VRC01 Prevents HIV-1 Transmission from Plasmacytoid Dendritic Cells to CD4 T Lymphocytes

Plasmacytoid dendritic cells (pDC) poorly replicate human immunodeficiency virus type 1 (HIV-1) but efficiently transfer HIV-1 to adjacent CD4 T lymphocytes. We found that coculture with T lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, and increases pDC maturation and alpha interferon (IFN-α) secretion. HIV-1 transfer to T lymphocytes is inhibited by broadly neutralizing antibody VRC01 with efficiency similar to that of cell-free infection of T lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-α secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition.     

Mycobacterium tuberculosis Promotes HIV trans-Infection and Suppresses Major Histocompatibility Complex Class II Antigen Processing by Dendritic Cells

Mycobacterium tuberculosis is a leading killer of HIV-infected individuals worldwide, particularly in sub-Saharan Africa, where it is responsible for up to 50% of HIV-related deaths. Infection by HIV predisposes individuals to M. tuberculosis infection, and coinfection accelerates the progression of both diseases. In contrast to most other opportunistic infections associated with HIV, an increased risk of M. tuberculosis infection occurs during early-stage HIV disease, long before CD4 T cell counts fall below critical levels. We hypothesized that M. tuberculosis infection contributes to HIV pathogenesis by interfering with dendritic cell (DC)-mediated immune control. DCs carry pathogens like M. tuberculosis and HIV from sites of infection into lymphoid tissues, where they process and present antigenic peptides to CD4 T cells. Paradoxically, DCs can also deliver infectious HIV to T cells without first becoming infected, a process known as trans-infection. Lipopolysaccharide (LPS)-activated DCs sequester HIV in pocketlike membrane invaginations that remain open to the cell surface, and individual virions are delivered from the pocket into T cells at the site of contact during trans-infection. Here we report that M. tuberculosis exposure increases HIV trans-infection and induces viral sequestration within surface-accessible compartments identical to those seen in LPS-stimulated DCs. At the same time, M. tuberculosis dramatically decreases the degradative processing and major histocompatibility complex class II (MHC-II) presentation of HIV antigens to CD4 T cells. Our data suggest that M. tuberculosis infection promotes a shift in the dynamic balance between antigen processing and intact virion presentation, favoring DC-mediated amplification of HIV infections.Dendritic cells (DCs) comprise a diverse family of cell types whose primary function is to initiate and drive immune responses. Myeloid DCs (myDCs) are essential antigen-presenting cells that monitor peripheral tissues for invading pathogens. myDCs bind and internalize bacteria and viruses using a variety of surface receptors. When stimulated by pathogenic or inflammatory signals, peripheral-tissue DCs migrate to lymphoid tissues and undergo maturation, degrading stored antigens into peptides that are loaded onto major histocompatibility complex class II (MHC-II) molecules and expressed on the cell surface for presentation to CD4 T cells (reviewed in reference 4). In addition to presentation of processed peptide antigens, DCs carry intact, unprocessed proteins and pathogens from peripheral tissues to lymph nodes, where they can be passed to other antigen-presenting cells to increase the breadth of the immune response (reviewed in reference 10).HIV can exploit the natural trafficking of DCs to establish and amplify infection of CD4 T cells. DCs efficiently transfer intact, infectious HIV to T cells during immune interactions through a process known as trans-infection (14). DCs trans-infect HIV by binding and concentrating the intact virus at the cellular interface, forming an “infectious synapse” that concentrates HIV receptors on the T cell to the same site (24). Importantly, trans-infection does not require productive infection of the DCs, which are not infected efficiently by HIV in vitro or in vivo (14). Immature DCs significantly enhance infection of T cells through trans-infection, and prior activation by cytokine or bacterial stimuli markedly increases infectious synapse formation and concomitant trans-infection (2, 24, 33).Worldwide, nearly one-third of HIV-infected people are coinfected with Mycobacterium tuberculosis, and active tuberculosis disease (TB) is the number one cause of death in HIV-infected people. Coinfected individuals are 30 times more likely to progress to active TB, which can in turn increase HIV replication and accelerate the progression to AIDS (35). The mechanisms by which coinfection with M. tuberculosis and HIV accelerates the progression of both diseases are poorly understood.Lung macrophages are the primary target of M. tuberculosis infection, and active disease is characterized by unconstrained replication in these cells. Dendritic cells can also be infected by M. tuberculosis, but M. tuberculosis growth is restricted due to a lack of nutrient access in the DC phagolysosomal structure in which it resides (20). Importantly, M. tuberculosis-infected DCs traffic between the infected lung and draining lymph nodes, bringing bacterial antigens into lymphoid tissues to initiate CD4 T cell responses essential for disease control (39).Others have established that M. tuberculosis binds to and is internalized by DCs via an interaction between the mycobacterial cell wall component mannosylated lipoarabinomannan (ManLAM) and the cell surface receptor DC-SIGN on dendritic cells (15). After ManLAM stimulation, DCs begin to secrete interleukin-10 (IL-10) and show defects in immunostimulatory functions (15). However, a more recent study suggests that ManLAM may not be solely responsible for these outcomes (1).Previously, it has been shown that lipopolysaccharide (LPS) potently stimulates HIV trans-infection of CD4 T cells by DCs (24, 33). Therefore, we reasoned that M. tuberculosis and its products might similarly stimulate DC trans-infection during active M. tuberculosis infections. Further, we hypothesized that DC activation by M. tuberculosis would result in downmodulation of processing and MHC-II presentation of newly bound HIV particles, shifting the balance away from immune control in favor of viral dissemination and pathogenesis.Here, we demonstrate that M. tuberculosis infection of DCs enhances HIV trans-infection mediated through surface-accessible, pocketlike invaginations of the plasma membrane. Increased HIV trans-infection is accompanied by decreased MHC-II processing and presentation of HIV antigens to CD4 T cells. Our results suggest one mechanism whereby M. tuberculosis infection can fuel HIV dissemination in coinfected individuals and at the same time decrease immune control of both HIV and M. tuberculosis infections.     

Presence of IgM Antibodies Which Sensitize HIV-1-Infected Cells to Cytolysis by Homologous Complement in Long-Term Survivors of HIV Infection

Although human cells are resistant to homologous human complement due to the presence of species-specific membrane inhibitors, a naturally occurring IgM antibody which recognizes an asialo-oligosaccharide can sensitize HIV-1-infected cells for complement-mediated cytolysis. Therefore, we investigated whether long-term survivors of HIV-1 infection harbor such antibodies in their sera. Thirty of 31 sera from HIV-1 seropositive hemophilia patients who have survived HIV-1 infection 10 years or more showed appreciable cytolytic activity, while only 2 sera of 10 seropositive patients presumed to have been infected with HIV-1 (due to sexual contact) more recently showed cytolytic activity. On the other hand, only 7 out of 43 sera from seronegative hemophilia patients showed cytolytic activity. Immunofluorescence staining for IgM on HIV-L -infected cells essentially correlated with the cytolytic capacity of the sera. Therefore, naturally occurring IgM antibodies and/or generated IgM antibodies reactive with the HIV-L -infected cells in patients might have been responsible for long-term survival due to complement-mediated immune cytolysis which may, in conjunction with cytotoxic T lymphocytes, synergistically suppress the infected cells in vivo. Therefore, the transfusion of such IgM antibodies could be effective for the treatment of HIV-L -infected individuals.     

Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.     

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo.