Combinations of KIR3DL1 and HLA-Bw4 alleles protect against HIV infection and/or disease progression. These combinations enhance NK cell responsiveness through the ontological process of education. However, educated KIR3DL1+ NK cells do not have enhanced degranulation upon direct recognition of autologous HIV-infected cells. Since antibody-dependent cellular cytotoxicity (ADCC) is associated with improved HIV infection outcomes and NK cells overcome inhibition through killer cell immunoglobulin-like receptors (KIR) to mediate ADCC, we hypothesized that KIR3DL1-educated NK cells mediate anti-HIV ADCC against autologous cells. A whole-blood flow cytometry assay was used to evaluate ADCC-induced activation of NK cells. This assay assessed activation (gamma interferon [IFN-γ] production and/or CD107a expression) of KIR3DL1+ and KIR3DL1− NK cells, from HLA-Bw4+ and HLA-Bw4− HIV-positive and HIV-negative individuals, in response to autologous HIV-specific ADCC targets. KIR3DL1+ NK cells were more functional than KIR3DL1− NK cells from HLA-Bw4+, but not HLA-Bw4−, healthy controls. In HIV-infected individuals, no differences in NK cell functionality were observed between KIR3DL1+ and KIR3DL1− NK cells in HLA-Bw4+ individuals, consistent with dysfunction of NK cells in the setting of HIV infection. Reflecting the partial normalization of NK cell responsiveness following initiation of antiretroviral therapy, a significant correlation was observed between the peripheral CD4+ T-lymphocyte counts in antiretroviral therapy-treated subjects and the functionality of NK cells. However, peripheral CD4+ T-lymphocyte counts were not correlated with an anti-HIV ADCC functional advantage in educated KIR3DL1+ NK cells. The abrogation of the functional advantage of educated NK cells may enhance HIV disease progression. Strategies to enhance the potency of NK cell-mediated ADCC may improve HIV therapies and vaccines.
Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1+) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4+ T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4+ T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4+ T cells. No correlation between ADCC and viral load, CD4+ T cell count, or neutralization of HIV-1SF162 or other primary viral isolates was detected. Sera pooled from clade B HIV-1+ individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.
Purpose of review
We summarize current information on Fc receptor-mediated anti-viral activities of antibodies. These activites include FcγR-mediated inhibtion and neutralization of HIV on antigen presenting cells (APCs), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated virus inhibition (ADCVI).
An FcγR-mediated mechanism that results in augmented neutralization and may render non-neutralizing antibodies inhibitory has been demonstrated in APC. ADCC antibody activity correlates inversely with HIV disease progression in humans, and higher vaccine-induced ADCC antibody responses are associated with lower acute SIV viremia levels in macaques. Following vaccination with rgp120, ADCVI antibody levels are higher among those with a lower rate of sexually acquired HIV infection. Non-neutralizing SIV immune serum that prevents infection of newborn macaques after oral challenge has potent ADCVI antibody activity. Abrogating the ability of the Fc segment of the broadly neutralizing monoclonal antibody IgG1b12 to bind to FcγRs and to mediate ADCVI substantially reduces IgG1b12’s protective effect in a SHIV vaginal challenge model.
Fc-FcγR interactions play a critical role in the biological function of antibody and are likely to be instrumental in preventing or modulating lentiviral infection. Exploiting antibody responses that depend on Fc-FcγR interactions may help widen the breadth and increase the potency of vaccine-induced antibody. Although the importance of generating optimal Fab-antigen interactions cannot be overestimated, improving Fc-FcγR interactions through adjuvants or other strategies provides another option for improving HIV vaccines and immunotherapies.
Antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cell-mediated virus inhibition (ADCVI); neutralization; Fcγ receptor (FcγR); HIV
Antibody-Dependent Cellular Cytotoxicity (ADCC) may assist in preventing HIV or delaying disease progression. Most prior studies have analyzed Env-specific ADCC responses. We hypothesized that effective ADCC-based immunity may target conserved internal viral proteins such as Pol. We analyzed the ability overlapping Pol peptides to induce activation of NK cells via ADCC. We prospectively studied ADCC responses in 83 HIV+ subjects followed for 3 years. Pol peptides were commonly targeted by ADCC responses in these chronically infected subjects (in 32 of the 83 subjects). However, Pol-specific ADCC responses declined over time and did not correlate with delayed HIV progression, measured by either baseline CD4 T cells, CD4 T cell loss over time, baseline viral load or the need to start antiretroviral therapy. Although Pol is frequently targeted by ADCC in HIV+ subjects, the strength or specificity of Pol-specific ADCC responses needs to be modulated to be effective in delaying HIV progression.
Antibody-dependent cellular cytotoxicity (ADCC) is of considerable interest as an immune response that may facilitate control of HIV infection. We studied ADCC responses prospectively in a cohort of 79 HIV+ subjects followed for a mean of 2.3 years without antiretroviral therapy. We used a novel assay of the ability of ADCC to activate NK cells, either from the same HIV+ subject or a healthy blood donor. We found ADCC responses to either gp140 Env protein or HIV peptide pools were common in HIV+ subjects when NK cells from the HIV+ subject were used, but did not correlate with markers of HIV disease progression. In contrast, ADCC responses to whole gp140 Env protein were strongly associated with a slower decline in CD4 T cell loss when healthy donor NK cells were used as effectors. Our data had implications for induction of the most effective ADCC responses by HIV vaccines.
HIV; ADCC; NK Cells; CD4 T cells; viral load
The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.
There is growing interest in HIV-specific antibody-dependent cellular cytotoxicity (ADCC) as an effective immune response to prevent or control HIV infection. ADCC relies on innate immune effector cells, particularly NK cells, to mediate control of virus-infected cells. The activation of NK cells (i.e., expression of cytokines and/or degranulation) by ADCC antibodies in serum is likely subject to the influence of other factors that are also present. We observed that the HIV-specific ADCC antibodies, within serum samples from a panel of HIV-infected individuals induced divergent activation profiles of NK cells from the same donor. Some serum samples primarily induced NK cell cytokine expression (i.e., IFNγ), some primarily initiated NK cell expression of a degranulation marker (CD107a) and others initiated a similar magnitude of responses across both effector functions. We therefore evaluated a number of HIV-relevant soluble factors for their influence on the activation of NK cells by HIV-specific ADCC antibodies. Key findings were that the cytokines IL-15 and IL-10 consistently enhanced the ability of NK cells to respond to HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate “educated” KIR3DL1+ NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate “uneducated” KIR3DL1+ NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies in vitro. Manipulating the immunological environment to enhance the potency of NK cell-mediated HIV-specific ADCC effector functions could be a promising immunotherapy or vaccine strategy.
Partial control of HIV occurs during acute infection, although the mechanisms responsible are poorly understood. We studied the ability of antibody-dependent cellular cytotoxicity (ADCC) antibodies in serum to activate natural killer (NK) cells in longitudinal samples from 8 subjects with well-defined early HIV infection who controlled viremia to low levels. NK cell activation by ADCC antibodies to gp140 Env proteins was detected in half of the subjects at the first time point studied, a mean of 111 d after the estimated time of infection. In contrast, ADCC-mediated NK cell activation in response to linear HIV peptides evolved more slowly, over the first 2 y of infection. Our studies suggest that HIV-specific ADCC responses to conformational epitopes occur early during acute HIV infection, and broaden to include linear epitopes over time. These findings have implications for the immune control of HIV.
Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.
Despite strong efforts to improve clinical outcome of ovarian cancer patients by conventional and targeted immuno-based therapies, the prognosis of advanced ovarian cancer is still poor. Natural killer (NK) cells mediate antibody-dependent cellular cytotoxicity (ADCC), release immunostimulatory cytokines and thus function as potent anti-tumour effector cells. However, tumour cells developed mechanisms to escape from an effective immune response. So highly immunogenic substances, like the 38 kDa-preparation of M. tuberculosis, PstS-1, are explored for their potential to enhance cancer-targeted immune responses. In this study we examined the modulation of different NK cell functions by accessory monocytes and PstS-1. We focussed on NK cell activation as well as natural and antibody-dependent cellular cytotoxicity directed against epidermal-growth-factor-receptor (EGFR)-positive ovarian cancer cell lines.
Activation, cytokine release and cytotoxicity of NK cells stimulated by monocytes and PstS-1 were determined by FACS-analysis, ELISA, Bioplex assay and quantitative polymerase-chain reaction (qPCR). Transwell assays were used to discriminate cell-cell contact-dependent from contact-independent mechanisms. Five ovarian cancer cell lines (A2780, IGROV-1, OVCAR-3, OVCAR-4 and SKOV-3) with different EGFR-expression were used as target cells for natural and antibody-dependent cellular cytotoxicity assays. Cetuximab (anti-EGFR-antibody) was used for ADCC studies.
Our data show that monocytes effectively enhance activation as well natural and antibody-dependent cytolytic activity of NK cells. PstS-1 directly stimulated monocytes and further activated monocyte-NK-co-cultures. However, PstS-1 did not directly influence purified NK cells and did also not affect natural and antibody-dependent cellular cytotoxicity directed against EGFR-positive ovarian cancer cells, even in presence of monocytes. Direct cell-cell contact between NK cells and monocytes was required for NK activation, while released cytokines seemed to play a minor role.
Our data suggest that monocytes enhance natural and antibody-dependent cytotoxic activity of NK cells in a cell-cell contact dependent manner. The TLR-agonist PstS-1 provides additional monocyte activation and induces NK activation markers, while NK cytotoxicity remains unaffected. We conclude that monocytes provide accessory function for ADCC exerted by NK during antibody-based cancer immunotherapy directed against EGFR-positive ovarian cancer cells.
NK cell; PstS-1; Ovarian cancer; BCG; Immunotherapy; Cetuximab
The humoral immune response of the human host against the human immunodeficiency virus (HIV) type 1 (HIV-1) envelope glycoproteins comprises virus-neutralizing antibodies (NAs), antibody-dependent cellular cytotoxicity-mediating (ADCC) antibodies, and infection-enhancing antibodies (IEAs). Because of their potential significance for the outcome of infection with this virus, we have studied the relative prevalence of NAs, ADCC antibodies, and IEAs in the sera of patients infected with HIV. Our results demonstrate that while ≥60% of serum samples are positive for NAs or ADCC antibodies, 72% of these serum samples mediate the enhancement of infection in the presence of complement. In patients with low CD4 counts, NA and ADCC antibody levels tend to decrease, while IEA levels increase. A significant positive correlation was found only between the presence of ADCC antibodies and the presence of antibodies that neutralized HIV-1 in the presence of complement. These results show that the anti-HIV-1 humoral immune response consists of a mixture of antibodies that may inhibit or enhance HIV infection and whose ratios may vary in different stages of the infection.
Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.
With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.
Several studies have shown that cytotoxic T lymphocytes (CTLs) play an important role in controlling HIV/SIV infection. Notably, the observation of escape mutants suggests a selective pressure induced by the CTL response. However, it remains difficult to assess the definite role of the cellular immune response. We devise a computational model of HIV/SIV infection having a broad cellular immune response targeting different viral epitopes. The CTL clones are stimulated by viral antigen and interact with the virus population through cytotoxic killing of infected cells. Consequently, the virus population reacts through the acquisition of CTL escape mutations. Our model provides realistic virus dynamics and describes several experimental observations. We postulate that inter-clonal competition and immunodominance may be critical factors determining the sequential emergence of escapes. We show that even though the total killing induced by the CTL response can be high, escape rates against a single CTL clone are often slow and difficult to estimate from infrequent sequence measurements. Finally, our simulations show that a higher degree of immunodominance leads to more frequent escape with a reduced control of viral replication but a substantially impaired replicative capacity of the virus. This result suggests two strategies for vaccine design: Vaccines inducing a broad CTL response should decrease the viral load, whereas vaccines stimulating a narrow but dominant CTL response are likely to induce escape but may dramatically reduce the replicative capacity of the virus.
As a result of their high mutation rate, HIV and its counterpart SIV in non-human primates can evade recognition by the host immune response through the generation of viral variants, the so-called escape mutants. This avoidance of cytotoxic T lymphocyte (CTL) mediated killing seems to be one of the major reasons why virus replication is not controlled effectively. However, it remains difficult to investigate the critical properties of the dynamics of immune escape. To this end, we developed a new computational model of HIV/SIV infection consisting of several CTL clones that can recognize specific parts of viral proteins, i.e., epitopes. The simulations allow us to follow the dynamics of immune escape in detail and help to interpret longitudinal data of HIV/SIV infections. Interestingly, changing the relative sizes of the CTL clones leads to a different evolution of the virus. Instead of reducing the number of infected cells, an alternative strategy of vaccine design could be to reduce the replicative capacity of the virus that might have implications for disease progression.
Our research, inspired by the pioneering works of Isaac Witz in the 1980s, established that 40% of human metastatic melanomas express ectopically inhibitory Fc gamma receptors (FcγRIIB), while they are detected on less than 5% of primary cutaneous melanoma and not on melanocytes. We demonstrated that these tumoral FcγRIIB act as decoy receptors that bind the Fc portion of antimelanoma IgG, which may prevent Fc recognition by the effector cells of the immune system and allow the metastatic melanoma to escape the humoral/natural immune response. The FcγRIIB is able to inhibit the ADCC (antibody dependent cell cytotoxicity) in vitro. Interestingly, the percentage of melanoma expressing the FcγRIIB is high (70%) in organs like the liver, which is rich in patrolling NK (natural killer) cells that exercise their antitumoral activity by ADCC. We found that this tumoral FcγRIIB is fully functional and that its inhibitory potential can be triggered depending on the specificity of the anti-tumor antibody with which it interacts.
Together these observations elucidate how metastatic melanomas interact with and potentially evade humoral immunity and provide direction for the improvement of anti-melanoma monoclonal antibody therapy.
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.
Increasing evidence suggests that NK cells not only are critical in the initial host defense against pathogens but also may contribute to continued protection from human immunodeficiency virus type 1 (HIV-1) disease progression. NK cell cytolysis can be induced directly through diverse receptor families or can be induced indirectly through Fc receptors by antibodies mediating antibody-dependent cellular cytotoxicity (ADCC). ADCC has been implicated in both protection from simian immunodeficiency virus infection and slower progression of HIV-1 disease. ADCC activity declines with advancing infection, and yet the underlying mechanism for this dysfunction has not been defined, nor has it been determined whether the activity can be reconstituted. Here we demonstrate that NK cell-mediated ADCC is severely compromised in chronic HIV infection. The potency of ADCC function was directly correlated with baseline FcγRIIIa receptor (CD16) expression on NK cells. CD16 expression was negatively influenced by elevated expression of a group of enzymes, the matrix metalloproteinases (MMPs), normally involved in tissue/receptor remodeling. Inhibition of MMPs resulted in increased CD16 expression and augmented ADCC activity in response to antibody-coated target cells. These data suggest that MMP inhibitors may improve NK cell-mediated ADCC, which may provide subjects with an opportunity to harness the cytolytic power of NK cells through naturally occurring nonneutralizing HIV-specific antibodies.
Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIVmac251 challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIVmac251 stock had high levels of viral replication and rapid CTL escape. Unvaccinated naïve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of naïve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.
The role of Antibody-dependent cellular cytotoxicity (ADCC) responses in HIV-1 controllers is still unclear due to the heterogeneity of these patients. We analyzed 67 HIV-1 controllers and found significantly higher levels of ADCC antibodies in controllers versus viremic subjects (p = 0.017). Moreover, multivariate analysis revealed significantly higher ADCC titers in HLA B57- controllers compared to HLA-B57+ ones (p = 0.0086). These data suggest a role for ADCC in immune control of HIV, especially in HLA B57 negative controllers.
In the current report, we compared the specificities of antibody responses in sera from volunteers enrolled in three US NIH-supported HIV vaccine trials using different immunization regimens. HIV-1 Env-specific binding antibody, neutralizing antibody, antibody-dependent cell-mediated cytotoxicity (ADCC), and profiles of antibody specificity were analyzed for human immune sera collected from vaccinees enrolled in the NIH HIV Vaccine Trial Network (HVTN) Study #041 (recombinant protein alone), HVTN Study #203 (poxviral vector prime-protein boost), and the DP6-001 study (DNA prime-protein boost). Vaccinees from HVTN Study #041 had the highest neutralizing antibody activities against the sensitive virus along with the highest binding antibody responses, particularly those directed toward the V3 loop. DP6-001 sera showed a higher frequency of positive neutralizing antibody activities against more resistant viral isolate with a significantly higher CD4 binding site (CD4bs) antibody response compared to both HVTN studies #041 and #203. No differences were found in CD4-induced (CD4i) antibody responses, ADCC activity, or complement activation by Env-specific antibody among these sera. Given recent renewed interest in realizing the importance of antibody responses for next generation HIV vaccine development, different antibody profiles shown in the current report, based on the analysis of a wide range of antibody parameters, provide critical biomarker information for the selection of HIV vaccines for more advanced human studies and, in particular, those that can elicit antibodies targeting conformational-sensitive and functionally conserved epitopes.
The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV89.6P replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as two weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by four weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R = -0.83, p 0.015), and ADCVI (R = -0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV89.6P variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV89.6 and virus levels (R = -0.72 p =0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV89.6P challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing FcγR-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.
Antibody-dependent cellular cytotoxicity (ADCC) is a potentially effective adaptive immune response to human immunodeficiency virus (HIV) infection. The study of ADCC responses has been hampered by the lack of simple methods to quantify these responses and map effective epitopes. We serendipitously observed that standard intracellular cytokine assays on fresh whole blood from a cohort of 26 HIV-infected subjects identified non-T lymphocytes expressing gamma interferon (IFN-γ) in response to overlapping linear peptides spanning HIV-1 proteins. The effector cells were CD3− CD4− CD8− CD14− CD2+ CD56+/− NK lymphocytes and degranulated granzyme B and perforin in response to antigen stimulation. Serum transfer assays demonstrated that the specific response was mediated by immunoglobulin G. Fresh blood samples from half of the HIV-infected cohort demonstrated robust HIV peptide-specific IFN-γ expression by NK cells, predominately to Env, Pol, and Vpu HIV-1 proteins. Responses were readily mapped to define minimal epitopes utilizing this assay. Antibody-dependent, HIV-specific NK cell recognition, involving components of both innate and adaptive immune systems, represents a potentially effective immune response to induce by vaccination.
Immune escape mutations selected by human leukocyte antigen class I-restricted CD8+ cytotoxic T lymphocytes (CTLs) can result in biologically and clinically relevant costs to HIV-1 replicative fitness. This phenomenon may be exploited to design an HIV-1 vaccine capable of stimulating effective CTL responses against highly conserved, mutationally constrained viral regions, where immune escape could occur only at substantial functional costs. Such a vaccine might ‘channel’ HIV-1 evolution towards a less-fit state, thus lowering viral load set points, attenuating the infection course and potentially reducing the risk of transmission. A major barrier to this approach, however, is the accumulation of immune escape variants at the population level, possibly leading to the loss of immunogenic CTL epitopes and diminished vaccine-induced cellular immune responses as the epidemic progresses. Here, we review the evidence supporting CTL-driven replicative defects in HIV-1 and consider the implications of this work for CTL-based vaccines designed to attenuate the infection course.
CD8+ T lymphocyte; CTL; HIV-1; HLA; human leukocyte antigen class I; immune escape; replication capacity; vaccine design; viral fitness
Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to the control of HIV/SIV infection. However, little is known about the ADCC function of natural killer (NK) cells in non-human primate model. Here we demonstrated that ADCC function of NK cells was significantly compromised in chronic SIV/SHIV infection, correlating closely with the expression of FcγRIIIa receptor (CD16) on NK cells. CD32, another class of IgG Fc receptors, was identified on NK cells with higher expression in the infected macaques and the blockade of CD32 impacted the ability of NK cells to respond to antibody-coated target cells. The inhibition of matrix metalloproteases (MMPs), a group of enzymes normally involved in tissue/receptor remodeling, could restore NK cell-mediated ADCC with increased CD16 expression on macaque NK cells. These data offer a clearer understanding of NK cell-mediated ADCC in rhesus macaques, which will allow us to evaluate the ADCC repertoire arising from preclinical vaccination studies in non-human primates and inform us in the future design of effective HIV vaccination strategies.
Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.
Immunoconjugates; Cancer vaccines; Immune response to cancer; Immunotherapy; Pancreatic cancer