Diverse Ab effector functions mediated by the Fc domain have been commonly associated with reduced risk of infection in a growing number of nonhuman primate and human clinical studies. This study evaluated the anti-HIV Ab effector activities in polyclonal serum samples from HIV-infected donors, VAX004 vaccine recipients, and healthy HIV-negative subjects using a variety of primary and cell line–based assays, including Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cell-mediated viral inhibition, and Ab-dependent cellular phagocytosis. Additional assay characterization was performed with a panel of Fc-engineered variants of mAb b12. The goal of this study was to characterize different effector functions in the study samples and identify assays that might most comprehensively and dependably capture Fc-mediated Ab functions mediated by different effector cell types and against different viral targets. Deployment of such assays may facilitate assessment of functionally unique humoral responses and contribute to identification of correlates of protection with potential mechanistic significance in future HIV vaccine studies. Multivariate and correlative comparisons identified a set of Ab-dependent cell-mediated viral inhibition and phagocytosis assays that captured different Ab activities and were distinct from a group of ADCC assays that showed a more similar response profile across polyclonal serum samples. The activities of a panel of b12 monoclonal Fc variants further identified distinctions among the ADCC assays. These results reveal the natural diversity of Fc-mediated Ab effector responses among vaccine recipients in the VAX004 trial and in HIV-infected subjects, and they point to the potential importance of polyfunctional Ab responses.

Antibodies can provide protection against viral infections through a broad array of different mechanisms, including neutralization, agglutination, phagocytosis, and lysis of infected cells. Results of an immune correlates analysis from the RV144 HIV vaccine efficacy trial suggest that nonneutralizing Abs may have contributed to a reduced risk of infection (1). Several subsequent RV144 follow-up studies have provided evidence to suggest a role for nonneutralizing, FcR-mediated Ab effector functions (2, 3), including Ab-dependent cellular cytotoxicity (ADCC) (46) and phagocytosis (2). Moreover, previous work has demonstrated that ADCC-inducing Abs are detectable in early HIV infection (7), are enriched in long-term HIV nonprogressors (8), are associated with better outcomes in mother-to-child transmission (9, 10), and correlate with enhanced HIV control (11, 12). In animal models, effector functions support the protective activity of neutralizing Abs (13, 14), can protect neonatal macaques from SIV challenge (15), and have been associated with vaccine-mediated protection in multiple studies (16, 17), suggesting that they may play an important role in providing both protection against infection and better outcomes after infection (reviewed in Refs. 18, 19).

Because FcγR-mediated activities include an array of different mechanisms, mediated by multiple, divergent effector cell types, it is exceedingly difficult to measure the entirety of these activities in a single assay. In recent years, a number of different effector functional assays have been developed to assess ADCC, Ab-dependent cell-mediated viral inhibition (ADCVI), and Ab-dependent cellular phagocytosis (ADCP) activities in HIV studies. However, unlike neutralization activities, these Fc-mediated Ab effector functions are less often included in the routine evaluation of the immunogenicity of HIV vaccine candidates. This exclusion is partly due to the developmental challenges associated with well-characterized Fc effector functional assays, a relative lack of information to prioritize among or differentiate between available assays, and questions as to which mechanisms may predominate in vivo.

To fill this knowledge gap, we systematically evaluated a number of available Fc effector functional assays using a common set of samples with possible differences in effector function. The main objective of this study was to identify assays that most comprehensively and dependably capture Fc-mediated Ab functions mediated by different effector cell types and against different viral targets. Ultimately, these results may be used to facilitate the understanding of distinctive aspects of the Fc-mediated immune responses with broad and highly functional antiviral activity.

Each laboratory was provided common reagents, as applicable, and performed assays on a total of 140 blinded IgG samples purified from treated chronically HIV-infected subjects (n = 31), untreated chronically HIV-infected subjects (n = 28), elite controllers (20) (n = 31; all of whom achieved control of HIV infection at plasma HIV RNA levels of <2000 copies/ml and had documented infection for at least 1 y), recipients of AIDSVAX B/B gp120 (n = 30) and placebo (n = 10) 2 wk after the fourth inoculation in the VAX004 efficacy trial (21), HIV-seronegative subjects (n = 10), and control Abs as described in Table I. Among the control Abs, a panel of Fc domain–mutated b12 mAb variants with divergent Fc receptor binding (22) (described in Table II) and four blinded replicates each of IVIG (purified IgG from a pool of plasmas from healthy, HIV-negative subjects) (23), HIVIG (purified IgG from a pool of plasmas from HIV-infected subjects presumed to be infected with clade B virus) (24), and HIVIG-C (purified IgG from a pool of plasmas from HIV-infected subjects confirmed to be infected with clade C virus) were evaluated. Serum Ab samples were purified via melon gel–based depletion of prevalent serum proteins such as albumin and the majority of IgA, while passively enriching IgG, as previously described (25). A common stock of BaL virus (Bal.LucR.T2A.ecto produced in 293T.17 cells) or recombinant BaL gp120 was used in each assay, as appropriate when possible. Similarly, a common stock of cryopreserved PBMCs heterozygous for the F/V158 FcγRIIIa allotype (26) obtained by leukapheresis was used unless otherwise specified. All studies were approved by appropriate local Institutional Review Boards and each subject gave written informed consent.

Table I.
Study samples
Sample GroupN
Infected  
 Chronically infected/treated 31 
 Chronically infected/untreated 28 
 Elite controller 31 
 Uninfected 10 
Vaccinee  
 VAX004 30 
 Placebo 10 
Control (replicates)  
 VIG 
 HIVIG 
 HIVIG-C 
Fc point mutants  
 b12 10 
Sample GroupN
Infected  
 Chronically infected/treated 31 
 Chronically infected/untreated 28 
 Elite controller 31 
 Uninfected 10 
Vaccinee  
 VAX004 30 
 Placebo 10 
Control (replicates)  
 VIG 
 HIVIG 
 HIVIG-C 
Fc point mutants  
 b12 10 
Table II.
b12 point mutants and their relative binding affinities (EC50) determined by ELISA
VariantFcγRIFcγRIIAFcγRIIIA
R292A 0.91 0.43 0.65 
D270E 0.71 0.42 0.69 
S239A 0.87 0.87 0.22 
S298A 0.84 0.52 1.31 
K338A 1.21 1.5 0.41 
S267G 1.05 2.99 0.21 
G236A 0.44 8.63 0.93 
I332E 1.21 3.19 7.32 
SD/IE 1.29 5.99 31 
SD/IE/AL 1.06 3.41 90 
SD/IE/GA 0.91 49 66 
WT 
VariantFcγRIFcγRIIAFcγRIIIA
R292A 0.91 0.43 0.65 
D270E 0.71 0.42 0.69 
S239A 0.87 0.87 0.22 
S298A 0.84 0.52 1.31 
K338A 1.21 1.5 0.41 
S267G 1.05 2.99 0.21 
G236A 0.44 8.63 0.93 
I332E 1.21 3.19 7.32 
SD/IE 1.29 5.99 31 
SD/IE/AL 1.06 3.41 90 
SD/IE/GA 0.91 49 66 
WT 

Reproduced with permission from the American Society for Microbiology, Moldt et al. (22).

Table III summarizes different attributes of the seven functional assays, described briefly below.

Table III.
Descriptions of the functional assays
AssayNameAssay DescriptionAssay ReadoutReference
BVADCC Bound virion ADCC Inhibition of infection of target cells at bound virion stage Percentage decrease in x2028;infected target cells (36
GTL ADCC ADCC assessed by granzyme release Induction of GrB and uptake by target cells Percentage Ag-coated target cells taking up GrB (27
RFADCC Rapid fluorescent ADCC Killing of sensitized target cells labeled with vital dyes Percentage dye loss (2, 30
LUC ADCC ADCC assessed by luminescence Reduction of virus-derived LUC activity in infected target cells Percentage reduction of virus-derived LUC activity in infected target cells (28
ADCVI ADCVI Inhibition of virus production by target cells Percentage decrease of p24 production by target cells (ELISA) (38
Virion phagocytosis Virion phagocytosis Phagocytosis of virus (HIV-1 US657) by effector cells (THP-1) Percentage cells taking up virus × MFI of positive cells (measured by flow) (13
ADCP ADCP Phagocytosis of Ag-coated beads by effector cells (THP-1) Percentage cells stained × MFI of positive cells (measured by flow) (39
AssayNameAssay DescriptionAssay ReadoutReference
BVADCC Bound virion ADCC Inhibition of infection of target cells at bound virion stage Percentage decrease in x2028;infected target cells (36
GTL ADCC ADCC assessed by granzyme release Induction of GrB and uptake by target cells Percentage Ag-coated target cells taking up GrB (27
RFADCC Rapid fluorescent ADCC Killing of sensitized target cells labeled with vital dyes Percentage dye loss (2, 30
LUC ADCC ADCC assessed by luminescence Reduction of virus-derived LUC activity in infected target cells Percentage reduction of virus-derived LUC activity in infected target cells (28
ADCVI ADCVI Inhibition of virus production by target cells Percentage decrease of p24 production by target cells (ELISA) (38
Virion phagocytosis Virion phagocytosis Phagocytosis of virus (HIV-1 US657) by effector cells (THP-1) Percentage cells taking up virus × MFI of positive cells (measured by flow) (13
ADCP ADCP Phagocytosis of Ag-coated beads by effector cells (THP-1) Percentage cells stained × MFI of positive cells (measured by flow) (39

GranToxiLux ADCC.

The GranToxiLux (GTL; from OncoImmunin) ADCC assay was conducted essentially as previously described (27). For this study, the CEM.NKRCCR5 cell line was coated with the HIV-1 recombinant gp120 representing the BaL isolate. The recombinant gp120 was provided by Dr. George Lewis to match the AT-2 inactivated virus used in a bound virion ADCC (BVADCC) assay (see below). These target cells were labeled with GTL, which becomes fluorescent upon cleavage by granzyme B (GrB). Upon recognition of the targets mediated by the anti-Env Ab–FcR interactions, the FcγR-bearing effector cells deliver GrB to the HIV-infected target cells. Once internalized, GrB cleaves the peptide and releases a fluorescent signal that can be identified by flow cytometry. The assay readout is the percentage of Ag-coated target cells, which take up GrB. Both peak activity and area under the titration curve (AUC) were used as assay data summary measures. The AUC was calculated as the integrated background-subtracted net activity over a range of dilutions using the trapezoidal method and was truncated above zero.

Luciferase ADCC.

The luciferase (LUC) ADCC assay used the HIV-1 infectious molecular clone (pNL-LucR.T2A-BaL.ecto, referred to as BaL.LucR.T2A.ecto in this study) expressing the BaL envelope (HIV Bal.LucR.T2A.ecto/293T/17; GenBank accession no. AY426110; https://www.ncbi.nlm.nih.gov/nuccore/AY426110) and the Renilla LUC reporter gene to infect the CEM.NKRCCR5 cell line used as target cells (28, 29). Whole PBMCs, obtained from cryopreserved leukapheresis samples, were used as effectors as previously reported (26). In this assay, IgG-containing samples were incubated with the target cells in the presence of the effector cells for 6 h. The assay readout is the percentage of specific killing based on the decrease of virus-derived LUC activity. Both peak activity and AUC were used as assay data summary measures.

Rapid fluorescent ADCC.

A modification of the rapid fluorescent ADCC (RFADCC) assay (30) was used, as previously described (2). In brief, the CEM-NKR-CCR5 T cell line was labeled with intracellular CFSE and membrane dye PKH26 and then pulsed with recombinant SF162 gp120 protein. NK cells were enriched directly from fresh healthy donor whole blood using RosetteSep (Stemcell Technologies). Purified IgG was added to the CEM-NKr cells and incubated with NK cells for 4 h at 37°C. The cell mix was then fixed and the proportion of PKH26+ cells that lost intracellular CFSE (i.e., lysed target cells) was determined by FACS. Data presented represent activity at a test concentration of 100 μg/ml averaged over two replicates.

BVADCC.

Replication-defective but entry-competent AT-2 inactivated BaL virions (3133) were provided by Dr. Jeff Lifson (Frederick Cancer Research Institute, National Institutes of Health), permitting the detection of ADCC responses to epitopes that are exposed during viral entry (3436; as described in Refs. 35, 36). Briefly, CEM-NKr-CCR5+ target cells were sensitized by spinoculation (37) at 12°C that is nonpermissive for fusion, with a multiplicity of infection of ∼5. Target cells were then sensitized with Ab dilutions, washed to eliminate prozone effects, and used at 37°C in a 3-h RFADCC assay. The assay readout represents the percentage of cytotoxicity determined by dual dye loss from the target cells for each Ab dilution (30). Both peak activity and AUC were used as assay data summary measures. The AUC was calculated as the integrated control sample–normalized readout over a range of dilutions using the trapezoidal method and was truncated above zero.

ADCVI.

Briefly, the assay was performed as previously described (38) by coincubating HIV-infected target cells (CEM.NKR-CCR5 cells) with test Ab and fresh PBMCs at a 10:1 E:T ratio. After 7 d, virus yield was measured by p24 ELISA. Ab ADCVI activity was defined as the average percentage decrease of p24 production by target cells across p24 ELISA replicates.

Virion phagocytosis.

HIV-1US657 (GenBank U04908) virus was first incubated with test Ab and then added to THP-1 cells (American Type Culture Collection) as described (13). The percentage of FITC+ cells and their fluorescence intensity were determined by flow cytometry. The assay readout was determined by multiplying the percentage of FITC+ cells by the fluorescence intensity of positive cells. Appropriate controls were used to subtract background (internalization of FITC-labeled virus in the absence of Env-specific Ab). The average readout over replicates was determined and a sample’s response was defined as positive when the background-adjusted value was ≥116.

ADCP.

Briefly, recombinant SF162 gp120 (Immune Technology)–coated 1-μm fluorescent NeutrAvidin beads (Life Technologies) were incubated with purified IgG samples for 2 h at 37°C as previously described (39). THP-1 cells (American Type Culture Collection) were then added to the bead/Ab mix and incubated overnight to allow for phagocytosis, followed by fixation and analysis of bead uptake by FACS. Data presented represent activity at a test concentration of 100 μg/ml averaged over two replicates. The assay readout represents scaled integrated median fluorescence intensity (MFI) values (frequency × MFI). No prespecified positivity criteria were used.

Briefly, an array of selected HIV-1 Ags was printed in triplicate onto polylysine-functionalized glass slides using a robotic microarrayer (Arrayit). After blocking, arrays were incubated with samples containing the Abs of interest. For purified IgG, the dynamic range was found to be 50–300 ng/ml using VRC01. Dilution between 1:100 and 1:500 of serum samples allowed for detectable signal with minimal background. After incubation with Ab samples, arrays were blocked, washed, and probed for IgG signal using a fluorescently labeled goat anti-human IgG detection Ab, scanned on a GenePix 4200AL (Molecular Devices), and analyzed with GenePix Pro 6.0 (Molecular Devices) for the MFI of each spot.

All analyses were performed in R 3.1.1 (40).

Correlation analysis.

Spearman correlation coefficients were calculated using raw data and visualized in a two-panel R pairs plot. The scatter plots of each pair of assay readouts were displayed in the upper right. The correlation coefficients were displayed on a gradient color scale, overlaid by statistical significance stars for unadjusted p values from testing a zero correlation coefficient in the lower left.

Hierarchical clustering of functional assay data.

Ward’s method of hierarchical clustering was used to generate dendrograms that visualized correlations and groupings among functional assay readouts using the hclust() function in R (41). Integrated assay data were first mean centered and scaled by the SD across samples. One minus the Euclidean distances among assay readouts was used as the dissimilarity index in the clustering.

Principal components analysis.

To investigate the relationship between the functional assays and how Ab functions may differentiate different subject classes, principal component analysis (PCA) was performed on scaled, centered data using the prcomp() function in R. In PCA, the original activity measurements were algebraically converted and combined into a new set of uncorrelated composite variables, ranked in order of their contribution to explaining the variation in the data. These new variables are termed principal components (PCs). The first three PCs were visualized in a three-dimensional scatter plot produced using the scatterplot3d package in R (42). To minimize visual complexity of plots, two plots were produced: one to display PCA scores for individual subjects colored to identify subject class, the other to display coordinates of each functional assay on the projected coordinates. Points and text were colored to identify different functional assays on the triplot.

Abs purified from plasma samples from HIV-infected subjects, VAX004 participants, pooled IgG controls, and monoclonal b12 Fc variants were assessed in a series of blinded cell-based assays of Ab effector function (Tables I, II) . These assays included four measures of ADCC, two measures of ADCP, and one measure of ADCVI (Table III). These assays are based on different principles and measure various aspects of Ab effector function, as follows: 1) The GTL ADCC assay detects the presence of ADCC-mediating Abs using a flow-based assay to quantify the elimination of target cells that have been coated with recombinant HIV-1 gp120 or infected with HIV-1 (27). 2) The LUC ADCC assay measures the replication in target cells in the presence of effector PBMCs and IgG-containing samples of a recombinant HIV-1 provirus that stably expresses Renilla LUC (28, 29), yielding a readout of the percentage of specific killing of virus-infected cells. 3) The RFADCC assay utilizes costaining of target cells with a membrane dye and a viability dye before the addition of IgG-containing samples and effector cells, enabling quantification of target cell lysis. 4) The BVADCC is a modified version of the RFADCC assay, such that the target cells are sensitized by highly purified replication-defective but entry-competent AT-2 inactivated BaL virions (3133), permitting the detection of ADCC responses to epitopes that are exposed during viral entry (3436; as described in Refs. 35, 36). 5) The virion phagocytosis assay measures the ability of macrophages or monocytes to internalize Ab-coated FITC-labeled HIV (13). 6) The THP-1 ADCP assay uses an FcγR-expressing monocytic-like cell line to measure monocyte-mediated phagocytosis of IgG Ab-coated fluorescent beads (39). 7) The ADCVI assay is a measure of the ability of Ab, in combination with FcγR-bearing effector cells, to inhibit virus replication (38).

These different assay types were chosen because they have the potential to measure distinct Fc-mediated Ab effector functions or combinations thereof. For example, the ADCVI assay captures a cumulative measure of the role of Ab in reducing viral outgrowth. Thus, ADCVI can encompass ADCC, phagocytosis, and the production of cytokines/chemokines, all of which might be involved in the inhibition of virus outgrowth from infected cells. In contrast, the ADCC assays assess target cell killing, or effector cell degranulation, for example. The four ADCC assays exhibit a number of differences; the LUC ADCC and GTL ADCC assays use whole PBMC as effector cells, whereas the RFADCC assay uses an NK cell–enriched population obtained from whole blood. Thus, these assays might reveal differences in cytotoxicity mediated by NK cells versus other immune cells such as macrophages, neutrophils, and eosinophils. Another difference among the four ADCC assays is that the GTL ADCC, RFADCC, and BVADCC assays enable the analysis of target cell killing on the single cell versus the population level, because these assays are flow cytometry based. In contrast, the LUC ADCC assay provides a population-level readout of target cell killing. Some assays use infected target cells, whereas others coat target cells with gp120. Similarly, although the two phagocytosis assays (virion phagocytosis and ADCP) are similar, they also have an important difference. Specifically, the fluorescent beads used in the ADCP assay (0.1 μm) are ∼10-fold greater in diameter than a typical HIV-1 virion (0.1 μm) (43). Because phagocytosis is known to be impacted by particle size (44), the virion phagocytosis assay is important in that it measures phagocytosis of a highly physiologically relevant particle (i.e., a virion). However, the ADCP assay has unique advantages in that it does not involve the use of infectious virus and avoids all of the technical hurdles involved therein, and it is amenable to isolating the activity of responses toward individual Ags and epitopes.

Purified serum IgG was used to reduce the potential impact of other soluble factors known to modulate effector cell activity (45). Purified serum IgG from uninfected placebo recipients in VAX004 were used as additional controls for assay specificity. A uniform batch of BaL virus or recombinant BaL gp120 Ag, as dictated by assay format, was used to match the Abs evaluated across assays as much as possible. Additionally, unfractionated samples from a subset of the study cohort were also used for comparisons with purified serum IgG samples.

Replicated assessments of the activity of pooled polyclonal plasma IgG from healthy subjects (IVIG) and from HIV-infected subjects (HIVIG and HIVIG-C) were used to determine intra-assay reproducibility and the ability to distinguish positive from negative samples (Fig. 1). Generally, functional assays reliably differentiated positive from negative control samples, and the coefficients of variation (CV) among these blinded replicates ranged from 1 to 50% among positive controls, with an average of 13% across all assays. In most cases, wide CVs could be attributed to one of four replicates giving a dramatically different readout, rather than a broad distribution among all replicates. Variability up to 30% of the CV is often considered acceptable in functional assays such as those evaluated in the present study.

FIGURE 1.

Ab functional activity across subject groups and control samples. The functional activity of purified Abs from HIV-infected treated (Tx), untreated (Untx), or elite controllers (EC), Vax004 vaccinees (VAX004), and VAX004 placebo recipients (Placebo) was assessed across a suite of cell-based assays of Ab effector function, including ADCC activity (BVADCC, GTL ADCC, RFADCC, LUC ADCC), ADCVI, and phagocytic activity (virion phagocytosis, ADCP). Replicates of polyclonal IVIG, HIVIG, and HIVIG-C were used as negative and positive controls and to assess assay reproducibility, and they were evaluated at a different concentration (gray-shaded region). Activity magnitudes (y-axis scales) are described in the 2Materials and Methods and should not be compared across assays.

FIGURE 1.

Ab functional activity across subject groups and control samples. The functional activity of purified Abs from HIV-infected treated (Tx), untreated (Untx), or elite controllers (EC), Vax004 vaccinees (VAX004), and VAX004 placebo recipients (Placebo) was assessed across a suite of cell-based assays of Ab effector function, including ADCC activity (BVADCC, GTL ADCC, RFADCC, LUC ADCC), ADCVI, and phagocytic activity (virion phagocytosis, ADCP). Replicates of polyclonal IVIG, HIVIG, and HIVIG-C were used as negative and positive controls and to assess assay reproducibility, and they were evaluated at a different concentration (gray-shaded region). Activity magnitudes (y-axis scales) are described in the 2Materials and Methods and should not be compared across assays.

Close modal

Interestingly, HIVIG-C was observed to exhibit equivalent or somewhat lower activity relative to HIVIG in a number of assays, with the exception of phagocytosis activity detected by the ADCP assay, suggesting a distinction among assays. Notably, these control Ab samples were evaluated at a higher test concentration than those from infected and vaccinated subjects, and thus they should not be directly compared with the latter two sample sets. Additionally, we observed good reproducibility based on replicates performed on the same purified IgG samples or unfractionated samples in the ADCVI, virion phagocytosis, RFADCC, and ADCP assays (Supplemental Fig. 1), as well as apparent differentiation among positive and negative control samples, as previously reported in other blinded studies (1, 2, 12, 17).

We first examined the activity levels of polyclonal Abs purified from subjects who were HIV infected or vaccinated with AIDSVAX B/B gp120 and observed that they exhibited high intersubject and in some cases high intergroup variability (Fig. 1), indicating that Fc effector function responses likely differ between vaccinated and infected subjects and are probably affected by a multitude of factors such as host genetics, sex, and/or infection duration. We next compared activity levels among the different assays between HIV-infected subjects (treated, untreated, and elite controllers) versus HIV-uninfected subjects (VAX004 placebo recipients, whose serum demonstrated a lack of non–sp. act.) and did not observe high activity among the subject groups in the ADCVI, virion phagocytosis, ADCP, or LUC ADCC (AUC) assays. However, activity levels in the BVADCC (AUC), GTL ADCC (AUC), and RFADCC assays were generally higher in HIV-infected subjects than in HIV-uninfected subjects. Although many previous studies have reported potentiated or broadened Ab activity present among HIV controllers or long-term nonprogressors (7, 8, 4648), these findings, which differ from the present study in assays used and populations analyzed, were not confirmed in this study, consistent with a similar recent study where subjects were balanced for age, gender, and presence or absence of protective HLA class I alleles (49). We next narrowed the scope of this comparison with placebo versus vaccine participants in VAX004 and observed that placebo recipients exhibited little to no activity in most assays, whereas vaccine recipients exhibited higher activity than did placebo recipients in the BVA ADCC, GTL ADCC, and RFADCC assays, albeit reduced relative to that of infected subjects, consistent with the decreased prevalence of BaLgp120-reactive Abs in the vaccinated population (Supplemental Fig. 2). However, striking differences in activity were not observed between placebo and vaccine recipient samples in the LUC ADCC, ADCVI, virion phagocytosis, or ADCP assays. In a previous study in which the virus tested was more closely matched to the vaccine envelope, ADCVI activity was observed to be associated with reduced risk of infection in VAX004 recipients (12), suggesting that the relatively low response observed among vaccine recipients may result from our choice of virus strain. Overall, these findings indicate that the different assays used in this study generally did not capture redundant information, but instead captured relatively distinct immunological functions.

To more directly compare across functional assays, a correlation analysis for each pair of assays was performed across the cohort of 90 infected subjects. The functional activity of Abs from each infected subject was plotted across each pair of effector assays to identify whether individual subjects had responses of similar magnitude across assays, or whether some subjects possessed Abs with discrepant activity magnitudes, such as being highly phagocytic but with low ADCC potency (Fig. 2). Hierarchical clustering of all seven assay readouts based on the infected subjects further captured the relative similarity and dissimilarities between functional assessments. Relatively higher correlations were observed between summary values such as peak activity and AUC within a given assay when both measures were available (e.g., BVADCC peak activity versus BVADCC AUC). In contrast, low to moderate correlation was observed between different ADCC assays that used either virus or Ag-coated target cells, and no to very low correlations were observed for the other assays either among themselves or with the ADCC assays (e.g., ADCP versus ADCVI). This pattern was confirmed by the dendrogram, in which a number of ADCC assays (BVADCC, RFADCC, and GTL ADCC) clustered together in a single subclade on the right side of the dendrogram, whereas the LUC ADCC, ADCVI, virion phagocytosis, and ADCP assays clustered separately in individual subclades on the left side of the dendrogram. This analysis suggests that these assays capture distinct functional activities, as evidenced by the lower correlations among the readouts of these assays as compared with the more tightly clustered ADCC assays on the right side of the dendrogram.

FIGURE 2.

Functional correlations across HIV-infected subjects. Correlation plots presenting the activity of Abs from each subject across pairs of functional assays are shown. The direction and strength of Spearman correlation coefficients between functional assessments and their significance values (lower left), as well as the corresponding scatter plots (upper right), are presented. Correlation strength is represented via a color scale, where dark orange indicates perfect negative correlation and dark blue signifies perfect positive correlation. The correlation matrix is ordered based on hierarchical clustering. For assays in which multiple summary values were available (e.g., peak activity and AUC), both measurements are presented. Unadjusted p values are reported as *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Functional correlations across HIV-infected subjects. Correlation plots presenting the activity of Abs from each subject across pairs of functional assays are shown. The direction and strength of Spearman correlation coefficients between functional assessments and their significance values (lower left), as well as the corresponding scatter plots (upper right), are presented. Correlation strength is represented via a color scale, where dark orange indicates perfect negative correlation and dark blue signifies perfect positive correlation. The correlation matrix is ordered based on hierarchical clustering. For assays in which multiple summary values were available (e.g., peak activity and AUC), both measurements are presented. Unadjusted p values are reported as *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Because functional coordination across multiple assays may differ among subject groups (2, 49), the correlation analysis was repeated within each subject group (Fig. 3). This analysis indicated that a higher degree of functional correlation was present in elite controllers than any other group. As previously observed in VAX003 (2), Abs from the VAX004 subjects generally exhibited reduced functional correlation. Although the limited correlations between Ab activities across different assays could be driven by the generally lower activity observed among vaccinees, Abs from treated and untreated subjects, who generally exhibited higher activity, were similarly less coordinated.

FIGURE 3.

Functional correlations within subject groups. Correlation plots presenting scatter plots among subjects by class across each pair of functional assays, organized by hierarchical clustering (LUC ADCC AUC, BVADCC AUC, GTL ADCC AUC, RFADCC, ADCVI, virion phagocytosis, and ADCP), are shown. The strength of Spearman correlation coefficients is presented in a color scale, where dark orange indicates perfect negative correlation and dark blue is perfect positive correlation. Unadjusted p values are reported as *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Functional correlations within subject groups. Correlation plots presenting scatter plots among subjects by class across each pair of functional assays, organized by hierarchical clustering (LUC ADCC AUC, BVADCC AUC, GTL ADCC AUC, RFADCC, ADCVI, virion phagocytosis, and ADCP), are shown. The strength of Spearman correlation coefficients is presented in a color scale, where dark orange indicates perfect negative correlation and dark blue is perfect positive correlation. Unadjusted p values are reported as *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

To further assess the differences among subject groups and assays, a PCA of all functional assay readouts was conducted based on the cohort of 90 HIV-infected subjects and 30 VAX004 vaccine recipients. We found that the first three lead principal components of the assay readouts explained a total of 72% variation of the data (Fig. 4A), with GTL AUC and BVADCC peak activity contributing the most to the first PC (Fig. 4B). Consistent with the box plots presented in Fig. 1, subjects were generally well mixed across the lead PCs, with the exception of VAX004 subjects, which were distinguished from infected subjects by PC1 (Fig. 4A). Across assays, summary values (peak activity or AUC) from the same assay were again strongly grouped together across all three lead PCs. Similar to the clustering results, a number of ADCC assays differentiated themselves from ADCVI, ADCP, and phagocytosis assays in PC1. However, these grouped ADCC assays contributed to variation captured in PC2 and PC3 (Fig. 4B), which points toward distinctions among them in terms of the Ab activities that they capture. This analysis confirms similarities among the ADCC assays and also highlights distinctions between these Ab activities and those assessed in the ADCVI, virion phagocytosis, and ADCP assays. Similarly, it also recapitulates the absence of strong activity differences among the different groups of HIV-infected subjects.

FIGURE 4.

PC triplots. (A) Subjects are displayed as pinned dots in the three-dimensional space defined by the three lead PCs (representing 72% of the variation in the data). (B) Contributions of each assay to PC1, PC2, and PC3 are displayed as pinned dots.

FIGURE 4.

PC triplots. (A) Subjects are displayed as pinned dots in the three-dimensional space defined by the three lead PCs (representing 72% of the variation in the data). (B) Contributions of each assay to PC1, PC2, and PC3 are displayed as pinned dots.

Close modal

Lastly, the relative activity of b12 Fc domain point mutants (22) was assessed across assay types. These Ab variants were designed to have selectively increased or decreased binding affinities to the two major activating FcγR2s, particularly FcγR2a and FcγR3a (Table II, reproduced from Ref. 22). Although all Ab variants have been shown to bind Env-gp120 and neutralize HIV-1 virions as well as their wild-type counterpart, the function of an Ab variant in the ADCVI, ADCP, ADCC, and other similar assays was shown to be generally correlated with its FcγR binding ability (22, 39). Thus, we predicted that the b12 Ab variants with decreased FcγR affinity would have lower activity in these assays, whereas the affinity-enhanced b12 Ab variants would exhibit higher activity. Consistent with this hypothesis, the LUC ADCC, GTL ADCC, and the ADCP assays exhibited differentiation among variants, where affinity-enhanced mutants were generally associated with increased Ab activity, and affinity-compromised mutants were associated with reduced activity (Fig. 5). However, not all assays exhibited this predictable FcγR affinity–dependent activity in the context of the b12 panel. For example, no strong activity patterns related to FcγR binding affinity were observed in the ADCVI assay, which may reflect the fact that this assay measures multiple Ab functions simultaneously. Whereas FcγR2a affinity did not appear to be strongly associated with activity in the virion phagocytosis assay, the set of FcγR3a affinity-enhanced mutants demonstrated reduced activity. Additionally, FcγR2a and FcγR3a affinity mutants appeared to exhibit the opposite effect as might have been expected on BVADCC assay activity. Furthermore, the nature of the immune complexes formed are likely to be a key variable that may drive differences between monomeric Fc–FcγR interactions and observed activity in biological assays. Collectively, these observations point to the complexity of FcγR-mediated Ab activity, indicating that in many assays, activity is not a simple result of affinity for a single FcγR, but rather may reflect multiple and competing interactions, and that these differing dependencies provide further distinctions between the activities measured by each assay.

FIGURE 5.

Functional activities across a b12 Fc domain point mutant panel. Functional activities of b12 Fc point mutants with enhanced (dark color) or reduced (light color) binding to FcγRs are shown. Mutants are binned into groups representing those with altered binding to FcγR3a (green) or FcγR2a (blue). Checkered bars represent point mutants whose impact on binding affinity to FcγR3a opposes the impact on binding affinity to FcγR2a, or vice versa. RFADCC results are not shown because they were negative.

FIGURE 5.

Functional activities across a b12 Fc domain point mutant panel. Functional activities of b12 Fc point mutants with enhanced (dark color) or reduced (light color) binding to FcγRs are shown. Mutants are binned into groups representing those with altered binding to FcγR3a (green) or FcγR2a (blue). Checkered bars represent point mutants whose impact on binding affinity to FcγR3a opposes the impact on binding affinity to FcγR2a, or vice versa. RFADCC results are not shown because they were negative.

Close modal

Evidence has accumulated that beyond prevalence and magnitude of Ag-specific binding Abs, there are specific qualitative features of the humoral immune response other than virus neutralization that may contribute to reduced risk of HIV infection. A growing number of human and nonhuman primate studies have identified correlates of reduced risk of infection related to Ab effector function (reviewed in Ref. 18). Because these studies have often used diverse methods to characterize effector function, we sought to compare a set of cell-based assays of ADCC and phagocytic function for the purpose of identifying key similarities and differences between assays. Given the value of some of these assays to vaccine development based on their status as correlates of vaccine-mediated protection, this comparative study offers data to begin to consolidate the body of work that points toward extraneutralizing Ab activity as important to protection.

Although other studies have also sought to compare different assays of Fc receptor function, to our knowledge ours is the first to report systematic evaluation of multiple assays of Fc receptor–mediated anti–HIV-1 Ab effector function, based on a common set of active and control samples tested in a blinded fashion. ADCC, ADCVI, and phagocytosis functions measured by seven different assays in four laboratories were determined for purified IgG samples from HIV elite controllers, treated and untreated HIV-infected subjects, as well as VAX004 vaccine and placebo recipients. Ab activity was generally lower among the vaccine recipients than among HIV-infected subjects. Despite previous reports describing enhanced Ab effector function among subjects exhibiting viral control or classified as long-term nonprogressors (8), we did not observe statistically enhanced Ab activity among elite controllers in any assay, consistent with a recent report (49). Because our study used purified IgG rather than dilute serum or plasma, it is possible that other serum factors may contribute to the enhanced Ab activity observed in viral suppressors in previous studies. Alternatively, this specific cohort, or responses to the BaL isolate, may differ from those evaluated previously in a way that impacted study outcome. Lastly, because this was not a prospective study based on randomized subjects, differences, or lack thereof, the Ab activity observed may be due to factors other than infection status.

Based on evaluation of polyclonal control samples, all seven assays appeared to reasonably discriminate the negative from the positive controls, although the virion phagocytosis assay rendered the smallest dynamic range of responses. Differences in activity between samples from HIV-infected subject groups and those observed in healthy uninfected subjects (VAX004 placebo group) were most pronounced in Ab functions captured by the BVADCC, GTL ADCC, and RFADCC assays. Such discrimination was also observed between VAX004 vaccine recipients relative to placebo samples and is an indication that these assays possess superior signal-to-noise resolution, or that these activities were better elicited among vaccinees and HIV-infected subjects than the other functions evaluated. Given the ability of the LUC ADCC, ADCVI, and ADCP assays to measure activity among our control samples and in some cases correlate with efficacy outcomes in other studies (1, 2, 12, 17), the differences in clustering observed in the present study provide support for utilization of these assays in characterizing a broad spectrum of humoral responses to vaccination.

A major purpose of this study was to identify functional assays that captured unique biological activities. To this end, we observed that although a subset of ADCC assays was grouped in the PCA, assay readouts measuring other Fc functions were only moderately or weakly correlated with each other, suggesting that multiple assays are needed to measure the full spectrum of distinct Ab functions in HIV vaccine studies aimed at identifying correlates of protection against HIV infection.

Interestingly, the coordination of different Ab functions based on their correlations appeared to be highest among elite controllers and weaker among other infected subject groups and VAX004 vaccine recipients. Reduced functional coordination was also observed in a previous study of a similar vaccine regimen conducted in a different population (VAX003) (2), suggesting that this pattern may be vaccine specific and population-independent. A more coordinated and polyfunctional Ab response was observed among vaccinees in the RV144 trial (2), which demonstrated a moderate level of efficacy against HIV infection. Taken together, these observations suggest that more orchestrated Ab responses may contribute to maintenance of lower levels of HIV viremia for HIV in infected subjects and a reduction in risk of HIV infection in vaccinated subjects, whereas the lack of coordinated Ab activity may be one possible reason for the lack of significant efficacy of the VAX004.

Complex associations were observed between FcγR affinity–dependent b12 mutants and Ab activity. Consistent with previous reports, ADCC and ADCP (22, 39) assays exhibited differentiation among Fc variants, where affinity-enhanced mutants were generally associated with increased Ab activity, and affinity-compromised mutants were associated with reduced activity. However, strong activity patterns proportional to FcγR binding affinity were not observed in the ADCVI and BVADCC assays. Although FcγR2a affinity did not seem to be associated with virion phagocytosis activity, the set of FcγR3a affinity–enhanced mutants demonstrated reduced phagocytosis activity, and FcγR3a affinity–compromised mutants demonstrated enhanced phagocytic activity. This relationship may indicate a role for competition between FcγR receptors in the response to an opsonized virus or infected cells, and it points toward the potential importance of relative affinity differences between different receptors. Such relative differences have most frequently been described as the ratio between binding affinity to activating (A) versus inhibitory (I) receptors, or the A/I ratio (50), associated with a resulting difference in intracellular signaling via inhibitory or activating motifs (51). Our results are consistent with apparent competition among activating receptors.

In some cases, FcγR2a and FcγR3a affinity mutants appeared to exhibit the opposite effect to what might have been expected. For example, in the BVADCC assay high-affinity mutants demonstrated lower activity than did their low-affinity counterparts. Interestingly, the BVADCC assay is known to exhibit a striking prozone effect, in which higher concentrations of Ab often lead to lower activity within a certain concentration range. The high affinity of these variants may have driven differential prozone effects across the panel.

Additionally, the use of mAbs recognizing individual epitopes also exposes potential differences in the effect of epitope presentation or availability across assays. Among assays that use different infected cell populations, epitopes may be differentially present or prevalent. Beyond this consideration, several of the assays use gp120-coated target cells, for which the CD4 binding site recognized by b12 is likely to be occluded. In fact, activity of this mAb was not observed in the RFADCC assay. Overall, the diversity of relationships between FcγR affinity and Ab activity observed for the b12 Fc domain point mutant panel further support and extend our general observations regarding the complementary nature of many of the assays.

This study provides an extensive comparative data set for different FcγR-mediated assays evaluated with a common set of samples. Diverse FcγR-mediated Ab activity at different levels was detected by assays using different effector cell types via different mechanisms. PCA and correlation analyses identified assays that provided the most distinct measures of Ab effector function. The b12 point mutant panel further supported and extended differentiation between assays in terms of their sensitivity to epitope and Fc affinity for FcγR. The relative activity of the polyclonal control HIVIG and HIVIG-C samples was also different among assays. Whereas HIVIG possessed higher activity than did HIVIG-C in the BVADCC, GTL ADCC, and LUC ADCC assays, no difference was observed in the RFADCC, ADCVI, or phagocytosis assays, and the polyclonal control activity profiles were reversed in the ADCP assay. Furthermore, evaluation of multiple functional assessments in combination enabled determination of relative differences in the extent of functional coordination between subject groups.

Collectively, these data identify and support the utilization of a complementary set of functional assays in the evaluation of humoral responses to HIV vaccines. Continued consideration of the benefit that extraneutralizing properties may provide in vivo, and which cell-based assays may best identify Ab types and responses that are associated with protection or therapeutic benefit, may also be key to advancing prophylactic and therapeutic Abs clinically (reviewed in Ref. 52). Identification of orthogonal functional assays described by this data set may provide a basis for future vaccine studies in defining meaningful functional Ab activities as potential correlates of protection against HIV infection, and deployment of these functional assessments will facilitate identification of functionally unique humoral responses in future HIV vaccine research efforts.

We thank Dr. Rick Koup of the National Institutes of Health Vaccine Research Center and the Collaboration for AIDS Vaccine Discovery Comprehensive T Cell Vaccine Immune Monitoring Consortium for PBMCs, Dr. Christina Ochsenbauer of the University of Alabama at Birmigham for LucR BaL reporter virus used in the LUC ADCC assay, Global Solutions for Infectious Disease for samples from the VAX004 trial, the Ragon Institute of MGH, MIT, and Harvard for cohort samples, Dr. Dennis Burton of The Scripps Research Institute for the panel of b12 Fc mutants, and Dr. Jeff Lifson (National Cancer Institute) for AT-2 inactivated BaL virions. The following reagent was obtained through the National Institutes of Health AIDS Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, catalog no. 3957): HIVIG from NABI and National Heart, Lung, and Blood Institute. HIVIG-C was prepared by using pooled purified IgG from clade C–infected subjects from South African blood bank samples donated by Dr. Lynn Morris of the National Institute for Communicable Diseases. We thank Lindsay Carpp for editorial assistance in the revision of the manuscript.

This work was supported by The Collaboration for AIDS Vaccine Discovery Grants OPP1032144 (Comprehensive Antibody Vaccine Immune Monitoring Consortium to D.C.M., G.F., D.N.F., K.G., and H.G.), OPP1032817 (to G.A., C.B.-K., K.G.D., H.D.C., and M.E.A.), OPP1032317 (to Y. H., B.B., and L.H.), and OPP1033109 (to G.K.L.); by National Institutes of Health Grants 1R01AI102691 (to M.E.A.), 5R01Al080289-03 (to G.A.), R01AI090656 (to D.N.F.), U01-AI067854 (to J.C.K.), and R01AI087181 (to G.K.L.); and by the Virology and Genetic Sequencing cores of the University of Alabama at Birmingham Center for AIDS Research as supported by National Institutes of Health Grant P30-AI-27767.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ADCC

Ab-dependent cellular cytotoxicity

ADCP

Ab-dependent cellular phagocytosis

ADCVI

Ab-dependent cell-mediated viral inhibition

AUC

area under the titration curve

BVADCC

bound virion ADCC

CV

coefficient of variation

GrB

granzyme B

GTL

GranToxiLux

LUC

luciferase

MFI

median fluorescence intensity

PC

principal component

PCA

principal component analysis

RFADCC

rapid fluorescent ADCC.

1
Haynes
B. F.
,
Gilbert
P. B.
,
McElrath
M. J.
,
Zolla-Pazner
S.
,
Tomaras
G. D.
,
Alam
S. M.
,
Evans
D. T.
,
Montefiori
D. C.
,
Karnasuta
C.
,
Sutthent
R.
, et al
.
2012
.
Immune-correlates analysis of an HIV-1 vaccine efficacy trial.
N. Engl. J. Med.
366
:
1275
1286
.
2
Chung
A. W.
,
Ghebremichael
M.
,
Robinson
H.
,
Brown
E.
,
Choi
I.
,
Lane
S.
,
Dugast
A. S.
,
Schoen
M. K.
,
Rolland
M.
,
Suscovich
T. J.
, et al
.
2014
.
Polyfunctional Fc-effector profiles mediated by IgG subclass selection distinguish RV144 and VAX003 vaccines.
Sci. Transl. Med.
6
:
228ra38
.
3
Li
S. S.
,
Gilbert
P. B.
,
Tomaras
G. D.
,
Kijak
G.
,
Ferrari
G.
,
Thomas
R.
,
Pyo
C. W.
,
Zolla-Pazner
S.
,
Montefiori
D.
,
Liao
H. X.
, et al
.
2014
.
FCGR2C polymorphisms associate with HIV-1 vaccine protection in RV144 trial.
J. Clin. Invest.
124
:
3879
3890
.
4
Bonsignori
M.
,
Pollara
J.
,
Moody
M. A.
,
Alpert
M. D.
,
Chen
X.
,
Hwang
K. K.
,
Gilbert
P. B.
,
Huang
Y.
,
Gurley
T. C.
,
Kozink
D. M.
, et al
.
2012
.
Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene family.
J. Virol.
86
:
11521
11532
.
5
Liao
H. X.
,
Bonsignori
M.
,
Alam
S. M.
,
McLellan
J. S.
,
Tomaras
G. D.
,
Moody
M. A.
,
Kozink
D. M.
,
Hwang
K. K.
,
Chen
X.
,
Tsao
C. Y.
, et al
.
2013
.
Vaccine induction of antibodies against a structurally heterogeneous site of immune pressure within HIV-1 envelope protein variable regions 1 and 2.
Immunity
38
:
176
186
.
6
Tomaras
G. D.
,
Ferrari
G.
,
Shen
X.
,
Alam
S. M.
,
Liao
H. X.
,
Pollara
J.
,
Bonsignori
M.
,
Moody
M. A.
,
Fong
Y.
,
Chen
X.
, et al
.
2013
.
Vaccine-induced plasma IgA specific for the C1 region of the HIV-1 envelope blocks binding and effector function of IgG.
Proc. Natl. Acad. Sci. USA
110
:
9019
9024
.
7
Forthal
D. N.
,
Landucci
G.
,
Keenan
B.
.
2001
.
Relationship between antibody-dependent cellular cytotoxicity, plasma HIV type 1 RNA, and CD4+ lymphocyte count.
AIDS Res. Hum. Retroviruses
17
:
553
561
.
8
Baum
L. L.
,
Cassutt
K. J.
,
Knigge
K.
,
Khattri
R.
,
Margolick
J.
,
Rinaldo
C.
,
Kleeberger
C. A.
,
Nishanian
P.
,
Henrard
D. R.
,
Phair
J.
.
1996
.
HIV-1 gp120-specific antibody-dependent cell-mediated cytotoxicity correlates with rate of disease progression.
J. Immunol.
157
:
2168
2173
.
9
Mabuka
J.
,
Nduati
R.
,
Odem-Davis
K.
,
Peterson
D.
,
Overbaugh
J.
.
2012
.
HIV-specific antibodies capable of ADCC are common in breastmilk and are associated with reduced risk of transmission in women with high viral loads.
PLoS Pathog.
8
:
e1002739
.
10
Milligan
C.
,
Richardson
B. A.
,
John-Stewart
G.
,
Nduati
R.
,
Overbaugh
J.
.
2015
.
Passively acquired antibody-dependent cellular cytotoxicity (ADCC) activity in HIV-infected infants is associated with reduced mortality.
Cell Host Microbe
17
:
500
506
.
11
Ahmad
R.
,
Sindhu
S. T.
,
Toma
E.
,
Morisset
R.
,
Vincelette
J.
,
Menezes
J.
,
Ahmad
A.
.
2001
.
Evidence for a correlation between antibody-dependent cellular cytotoxicity-mediating anti-HIV-1 antibodies and prognostic predictors of HIV infection.
J. Clin. Immunol.
21
:
227
233
.
12
Forthal
D. N.
,
Gilbert
P. B.
,
Landucci
G.
,
Phan
T.
.
2007
.
Recombinant gp120 vaccine-induced antibodies inhibit clinical strains of HIV-1 in the presence of Fc receptor-bearing effector cells and correlate inversely with HIV infection rate.
J. Immunol.
178
:
6596
6603
.
13
Hessell
A. J.
,
Hangartner
L.
,
Hunter
M.
,
Havenith
C. E.
,
Beurskens
F. J.
,
Bakker
J. M.
,
Lanigan
C. M.
,
Landucci
G.
,
Forthal
D. N.
,
Parren
P. W.
, et al
.
2007
.
Fc receptor but not complement binding is important in antibody protection against HIV.
Nature
449
:
101
104
.
14
Bournazos
S.
,
Klein
F.
,
Pietzsch
J.
,
Seaman
M. S.
,
Nussenzweig
M. C.
,
Ravetch
J. V.
.
2014
.
Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity.
Cell
158
:
1243
1253
.
15
Florese
R. H.
,
Van Rompay
K. K.
,
Aldrich
K.
,
Forthal
D. N.
,
Landucci
G.
,
Mahalanabis
M.
,
Haigwood
N.
,
Venzon
D.
,
Kalyanaraman
V. S.
,
Marthas
M. L.
,
Robert-Guroff
M.
.
2006
.
Evaluation of passively transferred, nonneutralizing antibody-dependent cellular cytotoxicity-mediating IgG in protection of neonatal rhesus macaques against oral SIVmac251 challenge.
J. Immunol.
177
:
4028
4036
.
16
Barouch
D. H.
,
Alter
G.
,
Broge
T.
,
Linde
C.
,
Ackerman
M. E.
,
Brown
E. P.
,
Borducchi
E. N.
,
Smith
K. M.
,
Nkolola
J. P.
,
Liu
J.
, et al
.
2015
.
Protective efficacy of adenovirus/protein vaccines against SIV challenges in rhesus monkeys.
Science
349
:
320
324
.
17
Barouch
D. H.
,
Stephenson
K. E.
,
Borducchi
E. N.
,
Smith
K.
,
Stanley
K.
,
McNally
A. G.
,
Liu
J.
,
Abbink
P.
,
Maxfield
L. F.
,
Seaman
M. S.
, et al
.
2013
.
Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys.
Cell
155
:
531
539
.
18
Lewis
G. K.
2014
.
Role of Fc-mediated antibody function in protective immunity against HIV-1.
Immunology
142
:
46
57
.
19
Ackerman
M. E.
,
Alter
G.
.
2013
.
Opportunities to exploit non-neutralizing HIV-specific antibody activity.
Curr. HIV Res.
11
:
365
377
.
20
Pereyra
F.
,
Addo
M. M.
,
Kaufmann
D. E.
,
Liu
Y.
,
Miura
T.
,
Rathod
A.
,
Baker
B.
,
Trocha
A.
,
Rosenberg
R.
,
Mackey
E.
, et al
.
2008
.
Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy.
J. Infect. Dis.
197
:
563
571
.
21
Flynn
N. M.
,
Forthal
D. N.
,
Harro
C. D.
,
Judson
F. N.
,
Mayer
K. H.
,
Para
M. F.
rgp120 HIV Vaccine Study Group
.
2005
.
Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection.
J. Infect. Dis.
191
:
654
665
.
22
Moldt
B.
,
Schultz
N.
,
Dunlop
D. C.
,
Alpert
M. D.
,
Harvey
J. D.
,
Evans
D. T.
,
Poignard
P.
,
Hessell
A. J.
,
Burton
D. R.
.
2011
.
A panel of IgG1 b12 variants with selectively diminished or enhanced affinity for Fcγ receptors to define the role of effector functions in protection against HIV.
J. Virol.
85
:
10572
10581
.
23
Imbach
P.
,
Barandun
S.
,
d’Apuzzo
V.
,
Baumgartner
C.
,
Hirt
A.
,
Morell
A.
,
Rossi
E.
,
Schöni
M.
,
Vest
M.
,
Wagner
H. P.
.
1981
.
High-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood.
Lancet
1
:
1228
1231
.
24
Cummins
L. M.
,
Weinhold
K. J.
,
Matthews
T. J.
,
Langlois
A. J.
,
Perno
C. F.
,
Condie
R. M.
,
Allain
J. P.
.
1991
.
Preparation and characterization of an intravenous solution of IgG from human immunodeficiency virus-seropositive donors.
Blood
77
:
1111
1117
.
25
Ackerman
M. E.
,
Dugast
A. S.
,
McAndrew
E. G.
,
Tsoukas
S.
,
Licht
A. F.
,
Irvine
D. J.
,
Alter
G.
.
2013
.
Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcγR2a and FcγR2b.
J. Virol.
87
:
5468
5476
.
26
Sambor
A.
,
Garcia
A.
,
Berrong
M.
,
Pickeral
J.
,
Brown
S.
,
Rountree
W.
,
Sanchez
A.
,
Pollara
J.
,
Frahm
N.
,
Keinonen
S.
, et al
.
2014
.
Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials.
J. Immunol. Methods
409
:
107
116
.
27
Pollara
J.
,
Hart
L.
,
Brewer
F.
,
Pickeral
J.
,
Packard
B. Z.
,
Hoxie
J. A.
,
Komoriya
A.
,
Ochsenbauer
C.
,
Kappes
J. C.
,
Roederer
M.
, et al
.
2011
.
High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses
.
Cytometry A
79
:
603
612
.
28
Pollara
J.
,
Bonsignori
M.
,
Moody
M. A.
,
Liu
P.
,
Alam
S. M.
,
Hwang
K. K.
,
Gurley
T. C.
,
Kozink
D. M.
,
Armand
L. C.
,
Marshall
D. J.
, et al
.
2014
.
HIV-1 vaccine-induced C1 and V2 Env-specific antibodies synergize for increased antiviral activities.
J. Virol.
88
:
7715
7726
.
29
Edmonds
T. G.
,
Ding
H.
,
Yuan
X.
,
Wei
Q.
,
Smith
K. S.
,
Conway
J. A.
,
Wieczorek
L.
,
Brown
B.
,
Polonis
V.
,
West
J. T.
, et al
.
2010
.
Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.
Virology
408
:
1
13
.
30
Gómez-Román
V. R.
,
Florese
R. H.
,
Patterson
L. J.
,
Peng
B.
,
Venzon
D.
,
Aldrich
K.
,
Robert-Guroff
M.
.
2006
.
A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity.
J. Immunol. Methods
308
:
53
67
.
31
Rossio
J. L.
,
Esser
M. T.
,
Suryanarayana
K.
,
Schneider
D. K.
,
Bess
J. W.
 Jr.
,
Vasquez
G. M.
,
Wiltrout
T. A.
,
Chertova
E.
,
Grimes
M. K.
,
Sattentau
Q.
, et al
.
1998
.
Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins.
J. Virol.
72
:
7992
8001
.
32
Chertova
E.
,
Bess
J. W.
 Jr.
,
Crise
B. J.
,
Sowder
R. C.
 II
,
Schaden
T. M.
,
Hilburn
J. M.
,
Hoxie
J. A.
,
Benveniste
R. E.
,
Lifson
J. D.
,
Henderson
L. E.
,
Arthur
L. O.
.
2002
.
Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus.
J. Virol.
76
:
5315
5325
.
33
Chertova
E.
,
Crise
B. J.
,
Morcock
D. R.
,
Bess
J. W.
 Jr.
,
Henderson
L. E.
,
Lifson
J. D.
.
2003
.
Sites, mechanism of action and lack of reversibility of primate lentivirus inactivation by preferential covalent modification of virion internal proteins.
Curr. Mol. Med.
3
:
265
272
.
34
Mengistu
M.
,
Ray
K.
,
Lewis
G. K.
,
DeVico
A. L.
.
2015
.
Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells. [Published erratum appears in 2015 PLoS Pathog. 11: e1004990]
PLoS Pathog.
11
:
e1004772
.
35
Lewis
G. K.
,
Guan
Y.
,
Kamin-Lewis
R.
,
Sajadi
M.
,
Pazgier
M.
,
Devico
A. L.
.
2014
.
Epitope target structures of Fc-mediated effector function during HIV-1 acquisition.
Curr. Opin. HIV AIDS
9
:
263
270
.
36
Guan
Y.
,
Pazgier
M.
,
Sajadi
M. M.
,
Kamin-Lewis
R.
,
Al-Darmarki
S.
,
Flinko
R.
,
Lovo
E.
,
Wu
X.
,
Robinson
J. E.
,
Seaman
M. S.
, et al
.
2013
.
Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding.
Proc. Natl. Acad. Sci. USA
110
:
E69
E78
.
37
O’Doherty
U.
,
Swiggard
W. J.
,
Malim
M. H.
.
2000
.
Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.
J. Virol.
74
:
10074
10080
.
38
Moldt
B.
,
Shibata-Koyama
M.
,
Rakasz
E. G.
,
Schultz
N.
,
Kanda
Y.
,
Dunlop
D. C.
,
Finstad
S. L.
,
Jin
C.
,
Landucci
G.
,
Alpert
M. D.
, et al
.
2012
.
A nonfucosylated variant of the anti-HIV-1 monoclonal antibody b12 has enhanced FcγRIIIa-mediated antiviral activity in vitro but does not improve protection against mucosal SHIV challenge in macaques.
J. Virol.
86
:
6189
6196
.
39
Ackerman
M. E.
,
Moldt
B.
,
Wyatt
R. T.
,
Dugast
A. S.
,
McAndrew
E.
,
Tsoukas
S.
,
Jost
S.
,
Berger
C. T.
,
Sciaranghella
G.
,
Liu
Q.
, et al
.
2011
.
A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples.
J. Immunol. Methods
366
:
8
19
.
40
Team
R. C.
2012
.
R: A Language and Environment for Statistical Computing.
R Foundation for Statistical Computing
,
Vienna, Austria
.
41
Murtagh
F.
1983
.
A survey of recent advances in hiearchical clustering algorithms.
Comput. J.
26
:
354
359
.
42
Hastie
T.
,
Tibshirani
R.
,
Friedman
J. H.
.
2009
.
The Elements of Statistical Learning: Data Mining, Inference, and Prediction.
Springer
,
New York
.
43
Pornillos
O.
,
Ganser-Pornillos
B. K.
.
2014
.
HIV-1 virion structure
. In
Encyclopedia of AIDS.
Hope
T. J.
,
Stevenson
M.
,
Richman
D.
, eds.
Springer
,
New York
. p.
1
6
.
44
Champion
J. A.
,
Walker
A.
,
Mitragotri
S.
.
2008
.
Role of particle size in phagocytosis of polymeric microspheres.
Pharm. Res.
25
:
1815
1821
.
45
Wren
L.
,
Parsons
M. S.
,
Isitman
G.
,
Center
R. J.
,
Kelleher
A. D.
,
Stratov
I.
,
Bernard
N. F.
,
Kent
S. J.
.
2012
.
Influence of cytokines on HIV-specific antibody-dependent cellular cytotoxicity activation profile of natural killer cells.
PLoS One
7
:
e38580
.
46
Ackerman
M. E.
,
Crispin
M.
,
Yu
X.
,
Baruah
K.
,
Boesch
A. W.
,
Harvey
D. J.
,
Dugast
A. S.
,
Heizen
E. L.
,
Ercan
A.
,
Choi
I.
, et al
.
2013
.
Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity.
J. Clin. Invest.
123
:
2183
2192
.
47
Wren
L. H.
,
Chung
A. W.
,
Isitman
G.
,
Kelleher
A. D.
,
Parsons
M. S.
,
Amin
J.
,
Cooper
D. A.
,
Stratov
I.
,
Navis
M.
,
Kent
S. J.
ADCC study collaboration investigators
.
2013
.
Specific antibody-dependent cellular cytotoxicity responses associated with slow progression of HIV infection.
Immunology
138
:
116
123
.
48
Chung
A. W.
,
Navis
M.
,
Isitman
G.
,
Wren
L.
,
Silvers
J.
,
Amin
J.
,
Kent
S. J.
,
Stratov
I.
.
2011
.
Activation of NK cells by ADCC antibodies and HIV disease progression.
J. Acquir. Immune Defic. Syndr.
58
:
127
131
.
49
Ackerman
M. E.
,
Mikhailova
A.
,
Brown
E. P.
,
Dowell
K. G.
,
Walker
B. D.
,
Bailey-Kellogg
C.
,
Suscovich
T. J.
,
Alter
G.
.
2016
.
Polyfunctional HIV-specific antibody responses are associated with spontaneous HIV control.
PLoS Pathog.
12
:
e1005315
.
50
Nimmerjahn
F.
,
Ravetch
J. V.
.
2005
.
Divergent immunoglobulin g subclass activity through selective Fc receptor binding.
Science
310
:
1510
1512
.
51
Clynes
R.
,
Maizes
J. S.
,
Guinamard
R.
,
Ono
M.
,
Takai
T.
,
Ravetch
J. V.
.
1999
.
Modulation of immune complex-induced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors.
J. Exp. Med.
189
:
179
185
.
52
Boesch
A. W.
,
Alter
G.
,
Ackerman
M. E.
.
2015
.
Prospects for engineering HIV-specific antibodies for enhanced effector function and half-life.
Curr. Opin. HIV AIDS
10
:
160
169
.

The authors have no financial conflicts of interest.

Supplementary data