HIV-1 Env- and Vpu-Specific Antibody-Dependent Cellular Cytotoxicity Responses Associated with Elite Control of HIV.

Studying HIV-infected individuals who control HIV replication (elite controllers [ECs]) enables exploration of effective anti-HIV immunity. HIV Env-specific and non-Env-specific antibody-dependent cellular cytotoxicity (ADCC) may contribute to protection from progressive HIV infection, but the evidence is limited. We recruited 22 ECs and matched them with 44 viremic subjects. HIV Env- and Vpu-specific ADCC responses in sera were studied using a novel enzyme-linked immunosorbent assay (ELISA)-based dimeric recombinant soluble FcγRIIIa (rsFcγRIIIa)-binding assay, surface plasmon resonance, antibody-dependent natural killer (NK) cell activation assays, and ADCC-mediated killing assays. ECs had higher levels of HIV Env-specific antibodies capable of binding FcγRIIIa, activating NK cells, and mediating granzyme B activity (all P < 0.01) than viremic subjects. ECs also had higher levels of antibodies against a C-terminal 13-mer Vpu peptide capable of mediating FcγRIIIa binding and NK cell activation than viremic subjects (both P < 0.05). Our data associate Env-specific and Vpu epitope-specific ADCC in effective immune responses against HIV among ECs. Our findings have implications for understanding the role of ADCC in HIV control.IMPORTANCE Understanding immune responses associated with elite control of HIV may aid the development of immunotherapeutic and vaccine strategies for controlling HIV infection. Env is a major HIV protein target of functional antibody responses that are heightened in ECs. Interestingly, EC antibodies also target Vpu, an accessory protein crucial to HIV, which degrades CD4 and antagonizes tetherin. Antibodies specific to Vpu are a common feature of the immune response of ECs that may prove to be of functional importance to the design of improved ADCC-based immunotherapy and preventative HIV vaccines.

Anti-HIV antibody-dependent activation of NK cells impairs NKp46 expression.

There is much interest in the potential of Ab-dependent cellular cytotoxicity (ADCC) to slow disease progression following HIV infection. Despite several studies demonstrating a positive association between ADCC and slower disease progression, it is possible that continued stimulation of NK cells by ADCC during chronic HIV infection could render these cells dysfunctional. Indeed, activation of NK cells by ADCC results in matrix metalloproteinase-induced reductions in CD16 expression and activation refractory periods. In addition, ex vivo analyses of NK cells from HIV-infected individuals revealed other alterations in phenotype, such as decreased expression of the activating NKp46 receptor that is essential for NK-mediated antitumor responses and immunity from infection. Because NKp46 shares a signaling pathway with CD16, we hypothesized that activation-induced downregulation of both receptors could be controlled by a common mechanism. We found that activation of NK cells by anti-HIV or anti-CD16 Abs resulted in NKp46 downregulation. The addition of a matrix metalloproteinase inhibitor attenuated NKp46 downregulation following NK cell activation by anti-HIV Abs. Consequently, these results suggest that continued stimulation through CD16 has the potential to impair natural cytotoxicity via attenuation of NKp46-dependent signals.

Polymorphisms and interspecies differences of the activating and inhibitory FcγRII of Macaca nemestrina influence the binding of human IgG subclasses.

Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His(131)). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro(159) and Tyr(160) impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His(131) and Met(132). Significantly, both His(131) and Met(132) are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His(131). These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques.

Antibody-dependent effector functions against HIV decline in subjects receiving antiretroviral therapy.

BACKGROUND: Combination antiretroviral therapy (cART) effectively controls human immunodeficiency virus (HIV) infection but does not eliminate HIV, and lifelong treatment is therefore required. HIV-specific cytotoxic T lymphocyte (CTL) responses decline following cART initiation. Alterations in other HIV-specific immune responses that may assist in eliminating latent HIV infection, specifically antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP), are unclear. METHODS: A cohort of 49 cART-naive HIV-infected subjects from Thailand (mean baseline CD4 count, 188 cells/µL; mean viral load, 5.4 log10 copies/mL) was followed for 96 weeks after initiating cART. ADCC and ADP assays were performed using serum samples obtained at baseline and after 96 weeks of cART. RESULTS: A 35% reduction in HIV type 1 envelope (Env)-specific ADCC-mediated killing of target cells (P<.001) was observed after 96 weeks of cART. This was corroborated by a significant reduction in the ability of Env-specific ADCC antibodies to activate natural killer cells (P<.001). Significantly reduced ADP was also observed after 96 weeks of cART (P=.018). CONCLUSIONS: This longitudinal study showed that cART resulted in significant reductions of HIV-specific effector antibody responses, including ADCC and ADP. Therapeutic vaccines or other immunomodulatory approaches may be required to improve antibody-mediated control of HIV during cART.

Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity antibodies in the absence of neutralizing antibodies.

A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.

HIV-specific antibody-dependent phagocytosis matures during HIV infection.

Antibody-dependent phagocytosis (ADP) is a potentially important immune mechanism to clear HIV. How HIV-specific ADP responses mature during HIV infection or in response to vaccinations administered, including the partially successful RV144 HIV vaccine, is not known. We established a modified ADP assay to measure internalisation of HIV antibody (Ab)-opsonised targets using a specific hybridisation internalisation probe. Labelled beads were coated with both biotinylated HIV gp140 envelope protein and a fluorescent internalisation probe, opsonised with Abs and incubated with a monocytic cell line. The fluorescence derived from the fluorescent internalisation probe on surface-bound beads, but not from internalised beads, was quenched by the addition of a complementary quencher probe. HIV Env-specific ADP was measured in 31 subjects during primary infection and early chronic HIV infection. Although ADP responses were present early during HIV infection, a significant increase in ADP responses in all 31 subjects studied was detected (P<0.001). However, when we tested 30 HIV-negative human subjects immunised with the Canarypox/gp120 vaccine regimen (subjects from the RV144 trial) we did not detect HIV-specific ADP activity. In conclusion, a modified assay was developed to measure HIV-specific ADP. Enhanced ADP responses early in the course of HIV infection were observed but no ADP activity was detected following the vaccinations administered in the RV144 trial. Improved vaccine regimens may be needed to capitalise on ADP-mediated immunity against HIV.

Immune escape from HIV-specific antibody-dependent cellular cytotoxicity (ADCC) pressure.

Effective immunity to HIV is poorly understood. In particular, a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV is controversial. We hypothesized that significant pressure from HIV-specific ADCC would result in immune-escape variants. A series of ADCC epitopes in HIV-infected subjects to specific consensus strain HIV peptides were mapped using a flow cytometric assay for natural killer cell activation. We then compared the ADCC responses to the same peptide epitope derived from the concurrent HIV sequence(s) expressed in circulating virus. In 9 of 13 epitopes studied, ADCC antibodies were unable to recognize the concurrent HIV sequence. Our studies suggest ADCC responses apply significant immune pressure on the virus. This result has implications for the induction of ADCC responses by HIV vaccines.

Specific antibody-dependent cellular cytotoxicity responses associated with slow progression of HIV infection.

Antibody-dependent cellular cytotoxicity (ADCC) is potentially an effective adaptive immune response to HIV infection. However, little is understood about the role of ADCC in controlling chronic infection in the small number of long-term slow-progressors (LTSP) who maintain a relatively normal immunological state for prolonged periods of time. We analysed HIV-specific ADCC responses in sera from 139 HIV(+) subjects not on antiretroviral therapy. Sixty-five subjects were LTSP, who maintained a CD4 T-cell count > 500/µl for over 8 years after infection without antiretroviral therapy and 74 were non-LTSP individuals. The ADCC responses were measured using an natural killer cell activation assay to overlapping HIV peptides that allowed us to map ADCC epitopes. We found that although the magnitude of ADCC responses in the LTSP cohort were not higher and did not correlate with CD4 T-cell depletion rates, the LTSP cohort had significantly broader ADCC responses compared with the non-LTSP cohort. Specifically, regulatory/accessory HIV-1 proteins were targeted more frequently by LTSP. Indeed, three particular ADCC epitopes within the Vpu protein of HIV were recognized only by LTSP individuals. Our study provides evidence that broader ADCC responses may play a role in long-term control of HIV progression and suggests novel vaccine targets.

HIV infection abrogates the functional advantage of natural killer cells educated through KIR3DL1/HLA-Bw4 interactions to mediate anti-HIV antibody-dependent cellular cytotoxicity.

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.

Rapid degranulation of NK cells following activation by HIV-specific antibodies.

Ab-dependent cellular cytotoxicity (ADCC) Abs stimulate NK cell effector functions and play a role in protecting from and controlling viral infections. We characterized ADCC Abs in a cross-sectional cohort of 80 HIV-infected subjects not on antiretroviral therapy. We analyzed ADCC response by killing fluorescently labeled target cells, as well as expression of IFN-gamma and the degranulation marker CD107a from activated NK cells as measured by a novel intracellular cytokine assay. HIV-specific ADCC directed toward Envelope proteins were present in the majority of 80 untreated HIV-infected individuals measured by killing function. Similarly, most subjects had HIV-specific Abs that mediated degranulation or cytokine expression by NK cells. Interestingly, there was a poor correlation between ADCC-mediated killing of fluorescently labeled whole Envelope protein-pulsed cell lines and Ab-mediated expression of IFN-gamma by NK cells. However, in contrast to healthy donor NK cells, autologous patient NK cells more effectively degranulated granzyme B in response to ADCC activation. Activation of NK cells in response to stimulation by HIV-specific Abs occurs at least as rapidly as activation of Gag-specific CTLs. Our studies highlight the complexity of ab-mediated NK cell activation in HIV infection, and suggest new avenues toward studying the utility of ADCC in controlling HIV infection.

Robust NK cell-mediated human immunodeficiency virus (HIV)-specific antibody-dependent responses in HIV-infected subjects.

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-gamma) 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-gamma 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.

Comparative evaluation of simian, simian-human, and human immunodeficiency virus infections in the pigtail macaque (Macaca nemestrina) model.

The global impact of HIV/AIDS intensifies the need for a preventive vaccine and nonhuman primate models can help provide critical insights into effective immunity. Pigtail macaques (Macaca nemestrina) are increasingly studied as a nonhuman primate model for AIDS. We compared the virologic and immunologic characteristics of HIV-1, SIV, and SHIV infection of naive pigtail macaques across a series of preclinical HIV vaccine studies. SIVmac251 and SIVmac239 infection of naive pigtail macaques resulted in a gradual decline in peripheral CD4+ T cells in the setting of high levels of viremia, approximating most closely human infection of HIV-1. In contrast, the CXCR4-utilizing SHIVmn229 virus resulted in rapid depletion of CD4+ T cells and minimal generation of humoral or cellular immune responses, similar to that observed with SHIV89.6P infection of rhesus macaques. Infection with the CCR5-utilizing, rhesus macaque passaged, SHIVSF162P3 resulted in some overall CD4+ T cell decline, however, three of eight macaques naturally control SHIVSF162P3 viremia to very low levels in the setting of robust adaptive immunity. Despite attempts at infecting pigtail macaques with HIV-1 strains passaged in juvenile pigtail macaques in vivo or in PBMC isolated from pigtail macaques in vitro, only lower nonsustained levels of viral replication were observed. Our results provide a series of virologic models with which to evaluate potential AIDS vaccines in pigtail macaques.

Short communication: characteristics of effective immune control of simian/human immunodeficiency virus in pigtail macaques.

Considerable evidence suggests both HIV-specific T cells and neutralizing antibodies (nAb) can, separately, assist control of viremia. T cell and nAb responses were studied in detail in three pigtail macaques protected from chronic simian/human immunodeficiency virus (SHIV) viremia by DNA prime/fowlpoxvirus boost vaccine regimens. Immunity was studied both after an initial intrarectal SHIV challenge, as well as during CD8 T cell depletion and a subsequent intravenous SHIV rechallenge. Remarkably, SHIV-specific CD4 and CD8 T cells were detectable in the absence of viremia following an initial SHIV challenge in one animal, subsequent to recovery from CD8 T cell depletion in all three animals, and following control of heterologous SHIV rechallenge in two animals. Neutralizing antibodies were also enhanced following CD8 depletion without recrudescence of viremia in all three animals. These observations, although in a small subset of animals, suggest the hypothesis that combinations of primed T cell immunity and neutralizing antibodies can maintain control of chronic primate lentiviral infections.

The role of HIV-specific antibody-dependent cellular cytotoxicity in HIV prevention and the influence of the HIV-1 Vpu protein.

There is growing interest in the role of anti-HIV antibody-dependent cellular cytotoxicity (ADCC) antibodies in the prevention and control of HIV infection. Passive transfer studies in macaques support a role for the Fc region of antibodies in assisting in the prevention of simian-human immunodeficiency virus (SHIV) infection. The Thai RV144 HIV-1 vaccine trial induced anti-HIV ADCC antibodies that may have played a role in the partial protection observed. Several observational studies support a role for ADCC antibodies in slowing HIV disease progression. However, HIV evolves to escape ADCC antibodies and chronic HIV infections causes dysfunction of effector cells such as natural killer (NK) cells that mediate the ADCC functions. Further, four recent studies show that the HIV-1 Vpu protein, by promoting release of virions, reduces the capacity of ADCC antibodies to recognize HIV-infected cells. The review dissects some of the recent research on HIV-specific ADCC antibodies and discusses mechanisms to further harness ADCC antibodies in the prevention and control of HIV infection.

Vaccines and vaccine strategies against HIV.

The HIV/AIDS pandemic is a global emergency and a preventive HIV vaccine is urgently needed. HIV has, however, proved a difficult pathogen to vaccinate against. This is largely because HIV has a very high mutation rate and can escape immune responses, it has a latent stage where it can rest silently integrated into host DNA, and neutralising antibodies that can neutralise diverse field strains have so far proved difficult to induce. There is however, considerable evidence now that HIV-specific CD4 and CD8 T cells can provide partial control of HIV replication and delay or prevent disease. Technologies to quantify and analyse HIV-specific T cells have advanced recently, and in particular ELISpot, intracellular cytokine staining and tetramer studies have provided clear analyses of the ability of HIV vaccines to induce T cell responses. The use of pools of overlapping HIV peptides as in vitro antigens has also provided a standardised reagent for accurate measurement of T cell responses. HIV protein vaccines have not induced broad neutralising antibodies or T cell responses and failed to protect humans in the only phase III efficacy trial yet completed. Viral vectors, such as canarypox, engineered to express HIV genes, have induced HIV-specific CD8 T cell responses in a minority of subjects in phase II trials and are proceeding to human efficacy trials. Currently, the most effective method of inducing CD8+ CTL immunity in non-human primates utilises priming with naked plasmid DNA and then boosting with recombinant viral vectors both encoding various parts of the HIV genome. Such vaccines have induced non-sterilising immunity to virulent Simian/Human immunodeficiency virus exposure in macaques and have entered phase I trials. Multiple other approaches are also being evaluated in what has become a global effort for a vaccine to prevent AIDS. Although an HIV vaccine is still a long way off, there is reason to be optimistic that a vaccine to prevent AIDS will eventually be developed.

Breadth of HIV-1 Env-specific antibody-dependent cellular cytotoxicity: relevance to global HIV vaccine design.

OBJECTIVE: The objective of this study is to determine the breadth of HIV-1 Env-specific antibody-dependent cellular cytotoxicity (ADCC) in HIV controllers and HIV progressors with a view to design globally relevant HIV vaccines. DESIGN: The breadth of ADCC towards four major HIV-1 Env subtypes was measured in vitro for 11 HIV controllers and 11 HIV progressors. METHODS: Plasma from 11 HIV controllers (including long-term slow progressors, viremic controllers, elite controller and posttreatment controller) and 11 HIV progressors, mostly infected with HIV-1 subtype B, was analysed for ADCC responses. ADCC assays were performed against 10 HIV-1 gp120 and 8 gp140 proteins from four major HIV-1 subtypes (A, B, C and E) and 3 glycosylation-mutant gp140 proteins. RESULTS: ADCC-mediated natural killer cell activation was significantly broader (P = 0.02) and of higher magnitude (P < 0.001) in HIV controllers than in HIV progressors. HIV controllers also showed significantly higher magnitude of ADCC-mediated killing of Env-coated target cells than HIV progressors to both HIV-1 subtype B and the heterologous subtype E gp140 (P = 0.001). We found good ADCC reactivity to subtype B and E Envs, less cross-reactivity to subtype A and minimal cross-reactivity to subtype C Envs. Glycosylation-dependent ADCC epitopes comprise a significant proportion of the total Env-specific ADCC response, as evident from the reduction in ADCC to nonglycosylated form of HIV-1 gp140 (P = 0.004). CONCLUSION: HIV controllers have robust ADCC responses that recognize a broad range of HIV-1 Env. Glycosylation of Env was found to be important for recognition of ADCC epitopes. Identifying conserved ADCC epitopes will assist in designing globally relevant ADCC-based HIV vaccines.

Azithromycin: more lethal than chloramphenicol?

Azithromycin is commonly used in sexual health and respiratory medicine, often when the diagnosis is presumptive. A recent article by Ray et al. reported that 1 out of 20?000 courses of low-dose azithromycin was associated with (sudden) cardiovascular death (including 1 out of 4000 courses in high-risk cardiovascular patients), ascribing these deaths to azithromycin itself. Here, we critique the actual study and examine conflicting data from randomised control trials, animal studies and observational data.

HIV-specific antibody immunity mediated through NK cells and monocytes.

The partial success of the RV144 trial re-energized the field of HIV vaccine research, which had stalled after vaccines based on neutralizing antibody and cytotoxic T cells had failed to induce protection. A large post-vaccine research effort has focused attention on the role of non-neutralizing antibodies in the protection afforded by the RV144 vaccine. These binding antibodies can initiate immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and combine elements of the adaptive and innate immune system in the form of antibodies and effector cells (including NK cells, monocytes and granulocytes). A complex interplay exists between the variable portion of the binding antibody and its HIV antigen target on one hand and the constant region of the antibody and the Fcγ-receptor of the effector cell on the other hand. Technical advances have revolutionized the abilities of scientist to detect the targets of non-neutralizing antibodies, including both envelope and non-envelope epitopes, and their role in forcing escape. Our understanding of the antibody characteristics (including IgG subclasses and Fc glycan profile) is providing valuable insights into their optimal structure and function. We expand on critical research on ADCC effector cells, particularly education of NK cells. We introduce the concept of HIV antibodydependent trogocytosis by monocytes as a potentially important aspect of HIV immunity. In summary, this review highlights recent advances in HIV-specific antibody immunity mediated through NK cells and monocytes.

Obstacles to ideal anti-HIV antibody-dependent cellular cytotoxicity responses.

A safe and effective vaccine against HIV is a global health priority. Large-scale phase III clinical vaccine trials based on neutralizing antibodies and cytotoxic T-lymphocytes have failed to provide protection, highlighting the lack of understanding of basic immune correlates of protection against HIV. The partial success of the RV144 vaccine trial, however, sparked an intense research effort to identify and describe the protective potential of non-neutralizing antibodies. Correlates of protection analyses have identified antibodies that induced antibody-dependent cellular cytotoxicity (ADCC) as potentially important. Despite the attractiveness of utilizing ADCC antibodies for HIV vaccine design, it is important to note that effective ADCC responses are contingent on many factors. As discussed in this review, these factors are important considerations for determining the feasibility of designing an optimal ADCC antibody-inducing vaccine construct. Important determinants of ADCC responses include characteristics of the antibody, such as isotype and subclass, antigen-specificity, titer, durability and glycosylation of the constant region. Second, ADCC immune responses are highly contingent on the natural killer (NK) cell effectors. This review will describe the current state of knowledge regarding the ontogeny of NK cells, highlighting the continuous "education" they undergo that determines their functional potential upon stimulation. Other important NK cell factors, such as constant region receptor polymorphisms, cellular exhaustion, and the effects of the cytokine milieu on cellular function, will also be covered. Finally, an exciting, but yet untested, role for NK cell-mediated ADCC lies in its potential ability to eliminate latently infected cells, which harbor the viral reservoir. The review will address the potential of a two-pronged attack, where latently infected cells are induced to express HIV antigens and then eliminated by NK cells via an ADCC mechanism, with the goal of inducing a cure.

Activation of NK cells by HIV-specific ADCC antibodies: role for granulocytes in expressing HIV-1 peptide epitopes.

HIV-specific ADCC antibodies could play a role in providing protective immunity. We have developed a whole blood ADCC assay that measures NK cell activation in response to HIV peptide epitopes. These HIV peptide-specific ADCC responses are associated with escape from immune recognition and slower progression of HIV infection and represent interesting HIV vaccine antigens. However, the mechanism by which these epitopes are expressed and whether or not they induce NK-mediated killing of cells expressing such peptide-antigens is not understood. Herein, we show that fluorescent-tagged ADCC peptide epitopes associate with blood granulocytes. The peptide-associated granulocytes become a specific target for antibody-mediated killing, as shown by enhanced expression of apoptosis marker Annexin and reduction in cell numbers. When HIV Envelope gp140 protein is utilized in the ADCC assay, we detected binding to its ligand, CD4. During the incubation, cells co-expressing gp140 and CD4 reduce in number. We also detected increasing Annexin expression in these cells. These data indicate that blood cells expressing HIV-specific ADCC epitopes are targeted for killing by NK cells in the presence of ADCC antibodies in HIV+ plasma and provide a clearer framework to evaluate these antigens as vaccine candidates.