Immunology and Cell Biology (2014), 1–9& 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14
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ORIGINAL ARTICLE

HIV-specific antibody-dependent phagocytosis
matures during HIV infection
Fernanda Ana-Sosa-Batiz1, Angus PR Johnston2,3, Haiyin Liu2, Robert J Center4, Supachai Rerks-Ngarm5,
Punnee Pitisuttithum6, Sorachai Nitayaphan7, Jaranit Kaewkungwal8, Jerome H Kim9, Nelson L Michael9,
Anthony D Kelleher10, Ivan Stratov1, Stephen J Kent1,11 and Marit Kramski1
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, wasquenched 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 (Po0.001). 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.
Immunology and Cell Biology advance online publication, 10 June 2014; doi:10.1038/icb.2014.42
Antibodies (Abs) are directed against multiple HIV epitopes1 andgenerally develop within a few weeks after infection. Neutralising Abs
directed against the HIV envelope protein can prevent HIV infection
in vitro and prevent infection of macaques with SIV/HIV Env
chimeric viruses.2–5 The development of neutralising Abs that areable to block infection of a broad range of HIV strains requires a long
Ab maturation process, which usually takes years to develop.6 HIV
broadly neutralising Abs are, however, difficult to elicit by standard
vaccination regimes, as they are unable to recapitulate the prolonged
presence of antigen required for generating highly affinity matured
broadly neutralising Abs.7
Although neutralising Abs are considered an efficient mode of Abmediated protection against HIV, Fc-mediated immune functions also
have an important role in protection.8,9 In contrast to the prolonged
development of broadly neutralising Abs, all HIV-infected individuals
rapidly develop non-neutralising Abs during the primary phase of
their infection. Abs can mediate different effector functions throughthe binding of their Fc region to the Fcg-receptor (FcgR), which

include antibody-dependent phagocytosis (ADP) and antibodydependent cellular cytotoxicity (ADCC).10,11
Numerous studies now support the role for ADCC activity in thecontrol of HIV infection where the presence of ADCC correlates witha better disease prognosis and slow HIV progression.12–17 In
comparison with neutralising Abs, ADCC Abs develop muchquicker and require lower levels of Ab maturation.18
In contrast to the growing body of literature on ADCC Abs,
comparatively little is known about ADP-mediating Abs in HIV
infection. ADP is a potentially important Ab effector function toeliminate immune complexes (that is, opsonised HIV) and HIVinfected cells. It is unknown when ADP Abs arise during HIV
infection and if they need a similarly long maturation process as
neutralising Abs. It was previously shown that ADP-mediating Abs
from controllers and untreated progressors exhibited similar ADP
activity, which was also elevated compared with subjects on antiretroviral therapy.19 However, little is known about ADP during early
HIV infection.

1Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Australia; 2Drug Delivery,
Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia; 3ARC Centre of Excellence in Convergent Bio-Nano Scienceand Technology, Monash University, Parkville, Australia; 4Burnet Institute, Melbourne, Australia; 5Department of Disease Control, Ministry of Public Health, Bangkok, Thailand;
6Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 7Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; 8Data Management Unit,
Mahidol University, Bangkok, Thailand; 9USA Military HIV Research Program, Walter Reed Army Institute of Research, Rockville, MD, USA; 10The Kirby Institute for Infection and
Immunity in Society, UNSW Medicine, Sydney, Australia and 11ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of Melbourne,
Melbourne, Australia
Correspondence: Professor SJ Kent, Department of Microbiology and Immunology, at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, 792
Elizabeth Street, Parkville, Victoria 3010, Australia.
E-mail: skent@unimelb.edu.au
Received 19 February 2014; revised 29 April 2014; accepted 29 April 2014

ADP in HIV
FA-S Batiz et al2

The role of ADP Abs in protection from HIV is also of growinginterest. Barouch et al.20 recently studied ADP in a macaquevaccination study using Adenovirus/poxvirus and adenovirus/
adenovirus vectors expressing HIV-1 mosaic antigens. Using this
vaccination strategy they demonstrated that protection was correlatedwith functional non-neutralising ADP Abs. However, this is the only
study that investigated the potential of vaccine-induced HIV-specific
ADP Abs in preventing HIV infection. In humans, the partially
protective RV144 vaccine elicited minimal HIV-specific neutralising
Abs but did induce non-neutralising Abs.21 Post hoc analyses
demonstrated that ADCC was associated with protection from HIV
acquisition in the RV144 trial22 and that serum IgA elicited by thevaccine interfered with binding and functional activity of ADCCmediating serum IgG.23 However, it is unknown if non-neutralising
Abs with ADP function were also induced by the RV144 vaccine
regimen.
To study the role played by Abs on clearance by phagocytosis ofvirus infected cells, it is necessary to use a robust method that is able
to evaluate ADP in vitro. The standard ADP assay developed byAckerman et al.24,25 assesses the phagocytic potential of Abs using Env
protein-coated fluorescent beads that are opsonised with purified IgG
and subsequently incubated with the phagocytic cell line THP1, which
expresses all three main FcgRs. A phagocytic score (% bead-positive
cells  mean fluorescence intensity) is calculated as an indicator of
ADP. However, this assay measures the sum of binding of beads onthe cell surface in addition to truly phagocytosed (that is, internalised)beads. To specifically assess the ability of Abs to assist internalisationof beads, we adapted a recent published method by Liu et al.26 to

study internalised beads. The assay involves adding a specifichybridisation internalisation probe (SHIP) that allows truly
phagocytised beads to be distinguished from surface-bound beads(Figure 1). We used this assay to investigate the development of ADPmediating Abs during the early stages of HIV infection and to
determine whether they can be induced by the RV144 preventative
vaccination strategy.
RESULTSADP-SHIP assay reliably discriminates between surface-bound andinternalised ADP targets
Non-neutralising Ab responses may assist in immunity to HIV andthere is a need to develop ADP assays to specifically measure HIV Abspecific phagocytosis. The standard ADP assay published24 utilises
beads with only one colour. Flow cytometry is used to determine the% of cells associated with beads, but it is not possible to distinguishbetween internalised beads and surface-bound beads (Figure 1a).
We developed a modified ADP assay, called ADP-SHIP, which
utilises both FITC and Cy5 (fluorescent internalisation probe, FIPCy5)
labelled beads. This dual labelling allows for the discrimination of
surface-bound and internalised beads after the addition of acomplementary quenching DNA sequence (QPC) at the end of theassay. The QPC cannot diffuse through the membrane of the cell and,
therefore, it only binds to FIPCy5 on beads bound to the cell surface
resulting in the quenching of the Cy5 fluorescence (Figure 1b).
Surface-bound and internalised beads are easily detected by flow
cytometry as FITC þ single-positive and FITC þ Cy5 þ doublepositive cells, respectively. All FITC þ cells are analysed for their

Standard Antibody-Dependent Phagocytosis Assay

Antibody-Dependent Phagocytosis SHIP Assay

Biotinylated gp140
Fc receptor

Fluorescent internalization
probe (FIPCy5)
Quencher probe (QPC)

Immunoglobulin G

Monocyte

Excitation wavelength

Fluorescent bead

Figure 1 Antibody-dependent phagocytosis (ADP) assays. (a) Standard ADP assay setup is shown as described in Ackerman et al.24 FITC-labelled beads arecoated with HIV envelope protein (Env) gp140 and incubated with HIV- or HIV þ purified IgG (left panel). The opsonised beads are then incubated with themonocytic cell line THP1 and ‘phagocytosed’ beads are identified by flow cytometry as FITC þ cells. This assay does not distinguish if beads are actuallyinternalised or if they only bind to the surface of the cell (middle and right panel). (b) The new ADP-SHIP assay is shown. FITC-labelled beads are coatedsimilar to the standard ADP assay, with HIV Env gp140 but also with a fluorescent internalisation probe (FIPCy5) and incubated with HIV  or HIV þ
purified IgG (left panel). The opsonised beads are then incubated with the monocytic cell line THP1 and ‘phagocytosed’ beads are measured by flow
cytometry as FITC þ Cy5 þ double-positive cells (middle panel). The addition of a complementary quenching probe (QPC) specifically blocks the Cy5
fluorescence derived from FIPCy5 on surface-bound beads, but not from internalised beads (right panel). Surface-bound beads are FITC þ single-positive andinternalised beads are double fluorescent (FITC þ Cy5 þ ).
Immunology and Cell Biology

ADP in HIV
FA-S Batiz et al3bead + cellsaBeads outside Beads Inside
37ºC

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ADP-SHIP

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Figure 2 Validation of the new ADP-SHIP assay. (a) Gating strategy for ADP-SHIP assay. The first two panels show gating strategy on live single cells. The
third panel shows gating on bead þ cells (i.e. cells associated with FITC þ beads, either internalised or on the surface). The fourth panel shows gating on
FITC þ Cy5 þ (internalised beads) and FITC þ Cy5- (surface associated beads) cells. The filled histogram shows a 4 1C-negative control where minimal
numbers of cells have internalised beads. The open histogram shows a sample incubated at 37 1C where 80.9% of cells associated with beads have at least
one internalised bead. (b) Equal numbers of bead þ cells for both the standard ADP and the ADP-SHIP assay was detected. (c) Deconvolution microscopyof ADP-mediated uptake. The left panel shows the standard ADP assay using FITC-labelled beads. The arrow for this panel shows beads associating withcells but does not discriminate whether the beads are surface bound or internalised. The right panel shows the ADP-SHIP assay. Arrows for this panel
indicate either surface-bound beads (FITC þ Cy5 ; green) or internalised beads (FITC þ Cy5 þ ; orange). The cell membrane was stained with wheat germ
agglutinin Alexa Flour 594 (blue). Complementary quenching probe: QPC.

Immunology and Cell Biology

ADP in HIV
FA-S Batiz et al
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ADP in HIV
FA-S Batiz et al
5

Cy5 fluorescence (gating shown in Figure 2a). Figure 2b shows thatthe additional binding of the FIPCy5 does not diminish the overall %of beads positive cells. This was independent of whether gp140 was
bound before, after, or simultaneously with FIPCy5 (Supplementary
Figure S1A). It further showed that for IgG from healthy donors the% of phagocytosed beads is overestimated as a proportion of thebeads that are only bound to the surface of the cells. One advantage isthat the ADP-SHIP assay directly determines the % of cells that haveinternalised beads (that is, are double-positive FITC þ FIPCy5). In
addition, the number of beads phagocytosed by each cell (one per cell,
two per cell, or more than two per cell) can be reliably determined.
Note that there is a low level of internalisation of gp140-coated beads
in the absence of HIV Abs, and thus the ADP-SHIP assay assesses theincrease in phagocytosis above this level (Figure 2b). Discriminationof surface-bound and internalised beads as seen by flow cytometry
was confirmed by deconvolution microscopy (Figure 2c).
Similar to the standard ADP assay, the ADP-SHIP assay needs
optimal conditions for each gp140 protein used including (i) gp140
protein concentration used for binding to the beads (Supplementary
Figure S1C), (ii) degree of gp140 biotinylation (Supplementary
Figures S1D and E) and (iii) IgG concentration (Supplementary
Figure S1C). These factors were carefully optimised (shown in
Supplementary Figure S1). The optimal levels of gp140 protein
coating were 0.5 mg ml 1 beads, the degree of biotinylation was
20-fold biotin excess and the concentration of IgG employed in theassay was 1 mg ml 1 . Coating of the beads with gp140 did not lead toan increase in binding of the beads to the surface of the target cell
(Supplementary Figure S1F).
To validate the new ADP-SHIP assay, we assessed ADP activity in
20 HIV-infected individuals and compared it with ADP activity of 20
healthy donors. Both the standard ADP and ADP-SHIP assays allowed
for discrimination between HIV þ and HIV  ADP activity. It isdifficult to directly compare these two assays because of the different
readouts; however, the difference in the ratio of phagocytosis of HIVpositive and HIV-negative samples was 2.08 for the standard ADP
assay and 2.04 for the ADP-SHIP assay (ratio of either the mean
phagocytic score or % FITC þ Cy5 þ cells of HIV þ /HIV  samples)(Figure 3a). However, we did note that the ADP-SHIP assay had awider dynamic range compared with the standard assay. There was nodifference in the levels of surface-bound gp140-coated beads between
the HIV  and HIV þ samples in the ADP-SHIP assay (Figure 3b).
The coefficient of variation of the ADP-SHIP and standard ADP assay
were similar at 4.56% and 7.24%, respectively across 10 HIV þ
replicates (Supplementary Figure S1G). When the number of beads
per cell was analysed, the ADP-SHIP assay revealed that the majorityof cells had only phagocytosed one bead per cell (mean: 14.1% of allcells; range 5.4–19.5%) but also a large proportion of one surfacebound bead per cell (mean: 6.3%; range 4.9–10%) was detected. A
smaller proportion of cells had two (mean 5.9%, range 1.2–9.8%) or
more than two beads per cell (mean 4.1%; range 0.47–8.7%)
phagocytosed (Figure 3d). Almost none of the cells had two or morethan two surface-bound beads per cell (mean: 1.1%; range 0.6–3.5%).

Overall, this equates to 24.1% of cells with phagocytosed beads,
whereas 7.4% of the cells have surface-bound beads. For this analysis
it is not necessary to use microscopy to detect the number ofinternalised beads. Through flow cytometry, it is possible to detect thewhole cell and not only slices through the cell, giving the possibility ofdetermining the number of beads internalised per cell.
ADP Abs during early HIV infection increase over time
Little is known about the development of ADP Abs during the courseof HIV infection. We, therefore, determined ADP in HIV-infectedpatients (n ¼ 31) who were not on treatment during primary andearly chronic infection. Serum samples were paired and median
duration of primary and early chronic infection was 4 months and 25
months, respectively. All subjects displayed ADP Abs at the early time
point, which was well above the ADP signal for HIV  subjects(Figure 4a). Over the 2-year–follow-up, ADP activity increased
significantly in 30 of the 31 individuals. For primary and earlychronic infection samples, there was a respective median±interquartile range ADP activity of 14.31%±7.20 and 25.19%±6.28% FITC þ
Cy5 þ cells. When the data were broken down by the number ofinternalised beads per cell (1, 2 or 42), we found an increase in ADP
across all subgroups in the early chronic infection samples(Figure 4b). There was a significant increase in the number of cellswith one internalised bead for early chronic infection samples(median: 15.61%; range: 11.54–20.55%) compared with primary
infection samples (median: 9.93%; range: 7.21–18.14%). The numberof cells with more than one bead internalised was also increased(median for primary infection samples: 4.59%; range: 2.85–13.16%;
median for early chronic infection samples: 9.82%; range:
5.37–19.10%). Cells with 1, 2 or 42 beads only bound to the surfaceof the cell and was independent of primary and early chronic
infection (Figure 4c). As there was a range of time points at which
the samples were collected (from 1–34 months after the estimatedtime of HIV infection), we plotted the duration of infection against
the level of ADP. We found ADP Abs were correlated with theduration of infection (Figure 4d).
No detectable ADP Abs in RV144 vaccinees
The ADP-SHIP assay was used to assess ADP activity in HIV
vaccinees of the RV144 trial21 in order to gain insight into whether
ADP can be elicited by preventative vaccines and to dissect theirpotential role in protection. Thirty samples from vaccine recipientsand 30 samples from placebo recipients were kindly provided by theThai-MHRP, all 2 weeks after the final vaccination (week 26 of thetrial). The IgG from plasma was first tested for ADP activity against
Env gp140 subtype B (AD8), which was one of the two subtypes used
for the protein boost used in the vaccination trial. Using the ADPSHIP assay, no significant difference between vaccinees and placebo
could be detected (Figure 5a). Vaccine and placebo samples from the
RV144 study did not show ADP activity above the HIV-negative
sample and were well below the HIV þ control samples. The RV144
vaccine regimen also employed a protein boost with a subtype A/E

Figure 3 Comparison of antibody-dependent phagocytosis with the standard ADP and the ADP-SHIP assays. (a) The phagocytic activity of 20 HIV-infected
and 20 healthy individuals was measured using purified IgG from plasma. Readout is as follows: standard ADP assay: phagocytic score (% beads-positive
cells  mean fluorescence intensity)/104) (left y axis); ADP-SHIP assay: % FITC þ and Cy5 þ cells (right y axis). The error bars represent mean ±s.e.m.
****P o0.0001 was determined using Student’s t-test. (b) Levels of surface-bound beads in the ADP-SHIP assay are similar for 20 HIV þ and 20
HIV  IgG samples. (c) Analysis of the number of beads per cell (internalised (FITC þ Cy5 þ ) vs surface bound (FITC þ Cy5 ) beads) using the ADP-SHIP
assay. In this assay, 29.7% of cells have at least one internalised bead (middle panel) and this can be broken down to cells with 1, 2 or 42 beads by
gating on the peaks as shown in the right panel. (d) Analysis of the numbers of internalised beads in the presence of 20 HIV þ sera. The error bars
represent mean ±s.e.m.
Immunology and Cell Biology

ADP in HIV
FA-S Batiz et al
6Internalised beads per cell40

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Figure 4 Development of ADP antibodies in HIV-infected patients during primary and early chronic infection. (a) ADP responses were evaluated in HIVinfected individuals (n ¼ 31) using paired serum samples. Median primary infection was 4 months and median early chronic infection was 2 years 1 month.
****P o0.0001 was determined using Wilcoxon matched-pairs signed rank test. (b) Analysis of the number of beads per cell comparing internalised(FITC þ Cy5 þ ) and (c) surface-bound (FITC þ Cy5-) beads in the presence of HIV Abs during primary and early chronic HIV infection (n ¼ 31). The error
bars represent median±IQR. ****Po0.05 was determined using Wilcoxon matched-pairs signed rank test. (d) Correlation of ADP responses with months
after HIV infection (n ¼ 31) R2 ¼ 0.55 and P o0.0001 determined using Pearson’s correlation.

Env protein. All samples were, therefore, also tested for their ADP
activity against Env gp140 subtype A/E 966, which was the same
subtype used for the vaccine DNA prime. No significant difference
could be detected in ADP activity between placebo and vaccine
Immunology and Cell Biology

recipients using either 966 or AD8 gp140 (Figure 5b). There were also
no differences in levels of surface-bound beads across the threegroups for either the subtype B or A/E gp140 protein-coated beads(Figures 5c and d).

ADP in HIV
FA-S Batiz et al
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Figure 5 Assessment of ADP in the RV144 HIV vaccine and placebo recipients. ADP was determined for 30 vaccine and 26 placebo recipients at week 26of the trial. As a positive control ADP for 15 HIV-infected subjects was measured. The grey dotted line indicates ADP for an HIV-negative subject. ADP wasmeasured against (a) Env gp140 subtype B (strain AD8) and (b) Env gp140 subtype A/E (strain 966). Surface-bound beads are shown for HIV-1AD8 andHIV-1966 gp140-coated beads in (c) and (d) respectively. The error bars represent mean±s.e.m.

DISCUSSION
There is limited knowledge about the potentially important role of
ADP Abs in the control or prevention of HIV infection. Such a role issupported by (i) the correlation of FcgR2a polymorphisms with HIV
disease progression,27 (ii) the correlation of the presence of
ADP-mediating IgG2 (binding more efficiently to FcgRIIa) withdelayed progression28,29 and (iii) HIV-associated downregulation of
FcgR2 expression in progressive infection leading to reduced ADP
activity.30
To standardise the measurement of ADP-mediating Abs Ackerman
et al.24 developed an ADP assay that employs a widely utilised
monocytic cell line that expresses a range of FcgRs, including both
inhibitory and activating FcgRs making it a highly useful assay tomeasure ADP function in both HIV infection and vaccine-mediatedprotection. We developed a modified assay, the ADP-SHIP assay,
which is able to specifically discriminate between surface-boundand internalised targets by flow cytometry without the need of
employing imaging techniques. Although the standard ADP and the
ADP-SHIP assay displayed comparable results, the dynamic range of

responses detected was greater for the ADP-SHIP assay. Further,
this assay can reliably detect the number of beads internalised
per cell and the proportion of target cells with only surface-boundbeads. It remains to be shown whether the ADP-SHIP assay offers
any improvement in assessing the functional significance of ADP in
HIV immunity over the standard ADP. Although we did not
find a difference in levels of surface-bound beads between HIV- andHIV þ samples, it is possible that future studies assessing cells at
different time points after exposing opsonised beads to the target
cells might reveal subtle differences. We also note that both thestandard ADP and the ADP-SHIP assays result in a level ofbackground association and internalisation of gp140-coated beads
in the absence of HIV Abs. Thus, both assays measure the Abdependent increase in phagocytosis (shown by comparing assays ofHIV compared with HIV þ IgG samples) rather than the total levelof phagocytosis.
Using the ADP-SHIP assay, we demonstrated that ADP-mediating
Abs develop early during infection and that ADP responses increaseover time in the absence of treatment. Whether this increase in ADP
Immunology and Cell Biology

ADP in HIV
FA-S Batiz et al
8

Table 1 Cohort characteristics for primary and early chronic HIV infection
Primary infection

Early chronic infection

Median CD4 count (range)

723 cells ml 1 (168–1220)

570 cells ml 1 (76–818)

Median HIV-viral load (range)
Median time after infection (range)

4.01 Log10 copies ml 1 plasma (2–5.88)

4.19 Log10 copies ml 1 plasma (1.69–5.32)
25 months (18–34)

4 months (1–11)

activity is due to the overall increase in ADP Ab titres or due to theoverall maturation of the ADP response (specificity and avidity)
remains to be elucidated. Ongoing studies from our own laboratory
have also shown that ADP responses are reduced by anti-retroviraltreatment (data not shown).
Inducing protective Ab responses through vaccination is a goal of
HIV vaccine studies. There is recent evidence on a potential role for
vaccine-induced ADP in protecting macaques from SIV infection.20
The only human HIV vaccine trial to date to show some success is the
RV144 trial, and we, therefore, analysed plasma samples from a subset
of vaccinees for ADP responses. We could not detect HIV Env-specific
ADP responses to either B or A/E subtype Env proteins following
vaccination. A recent publication shows a low level of ADP may be
induced by the RV144 regimen using the standard ADP assay.31
However, no ADP assays on sera from placebo-vaccinated or HIVinfected subjects were reported in that study. It will be important toanalyse further samples from RV144 trial vaccinated and placebo
subjects with more sensitive ADP assays in future studies. Although
speculative, it is possible that improved immunity to HIV may beachievable if vaccine regimens induce a broader range of nonneutralising Ab responses, including ADP.
To conclude, the new ADP-SHIP assay now provides a valuable andspecific tool to further study ADP-mediating Abs in the context ofvaccination and HIV infection.
METHODS
Patients and plasma samples
To establish the new ADP-SHIP assay, we studied serum samples from HIVinfected subjects (n ¼ 20) at various disease stages and from healthy subjects(n ¼ 20). Further longitudinal serum samples from patients (n ¼ 31) during
primary and early chronic infection (4.7 months after infection and 25.9
months after infection) were also assessed for their ADP activity. The list ofpatients and their clinical data (VL, CD4 count, time point of infection) ispresented in Table 1. To investigate ADP after vaccination serum samples ofrandomly selected vaccine recipients (n ¼ 30) and placebo recipients (n ¼ 26)of the RV144 vaccine trial21 were tested at week 26 of the trial. Informed
consent was obtained from all subjects, and the study was approved by therespective institutional ethics committees.
HIV-IG (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH:
Catalogue no. 3957 from NABI and NHLBI) was used as a positive control in
all ADP assays.

IgG purification from plasma
Total IgG was purified from 50–100 ml plasma using the Protein G HP
Multitrap and the Antibody Buffer Kit (both GE Healthcare, Buckinghamshire,UK) according to manufacturer’s instructions. Elution was repeated two times
and pooled elutes were washed twice with PBS using 30k Amicon Ultra-4
Centrifugal Filter Units (Millipore, Bedford, MA, USA). IgG concentration wasdetermined using the NanDrop 2000.

HIV Env gp140 protein and biotinylation
HIV Env gp140 protein (subtype B: AD8 clone of ADA; subtype A/E: 966,
clone 93TH966-89) was secreted by stably transfected HeLa cells and purified
by lentil–lectin affinity chromatography and size exclusion chromatography
Immunology and Cell Biology

using a 16/60 Superdex 200 column as previously described.32 Fractions
enriched for gp140 trimers were pooled and concentrated. Briefly, 20–200 mg of
gp140 Env was then biotinylated using EZ-Link Sulfo-NHS-LC-Biotin kit(Thermo Scientific, Rockford, IL, USA) according to the manufacture’s
instructions using a 20-fold biotin reagent excess. Excess biotin was removed
by washing twice with PBS using the 30 k Amicon Ultra-4 Centrifugal Filter
Units (Millipore).

Standard ADP assay
The standard ADP assay was performed as previously described.24 Briefly,
0.75 mg of biontinylated gp140 (unless stated otherwise gp140 AD8 was used)and 3 ml FITC-labelled NeutrAvidin FluoSpheres (beads 1 mm) (Invitrogen,
Carlsbad, CA, USA) were incubated together overnight at 4 1C andsubsequently washed with 2% BSA-PBS to remove unbound gp140. Ten
microliter of 1:10 diluted coated beads were opsonised with 1 mg ml 1 purified
human IgG, for 2 h at 37 1C before incubated with 1  10e5 THP1 cells(monocytic cell line, ATCC TIB-202) in a total volume of 200 ml. After an
incubation of 16 h at 37 1C cells were washed with PBS fixed in 1%
formaldehyde before acquired on a LSRII (BD Biosciences, San Jose, CA,
USA) and analysed using FlowJo analysis software version 9.6.2.

ADP-SHIP assay
The ADP-SHIP assay utilises a SHIP. Similar to the standard ADP, 0.75 mg
biotinylated HIV Env gp140 together with 3 ml of FITC-labelled NeutrAvidin
FluoSpheres (beads 1 mm) (Invitrogen) and 3 ml of 150 mM biotin- andCy5-labelled florescent internalisation probe (FIPCy5) (50 Cy5-TCAGTTCAGG
ACCCTCGGCT-N3 30 , Integrated DNA Technologies, Coralville, IA, USA)(26) were incubated overnight at 4 1C. Beads were then washed twice with 2%
BSA-PBS to remove unbound gp140 and FIPCy5. 10 ml of a 1:10 dilution ofcoated beads were opsonised with 1 mg ml 1 purified human IgG for 2 h at
37 1C, and then incubated with 1  10e5 THP1 cells in a total volume of
200 ml. After a 16 h incubation at 37 1C cells were washed with ice cold PBS andsurface-bound beads were quenched by adding 1.01 mg ml 1 of the complementary quenching probe (QPC) (50 -AGCCGAGGGTCCTGAACTGA-BHQ230 Integrated DNA Technologies) for 10 min at 4 1C.26 Cells were fixed in 1%
formaldehyde before acquired on the LSRII (BD) and analysed using
FlowJo analysis software version 9.6.2. The proportion of cells with surfacebound or internalised beads was determined by flow cytometry as shownin Figure 2a. The number of beads internalised (1, 2 or 42) was determinedby gating on the peaks associated with internalised beads as shown in
Figure 3c (right panel). There was a high degree of concordance of thisanalysis when we assessed 10 replicates of a HIV þ sera sample, with thecoefficient of variation of 1, 2, and 42 beads per cell being 3.89%, 3.62% and3.56%, respectively.

Deconvolution microscopy
Samples used for microscopy were treated the same way as described for the
ADP and ADP-SHIP assay with the exception that a membrane staining step
was performed before fixing. Briefly, 30 ml of 5 mg ml 1 of Wheat germ
agglutinin (Alexa Fluor594, Life Technologies, Carlsbad, CA, USA) was added
to the cells and incubated for 15 min at 4 1C. Cells were subsequently washedwith PBS and fixed in 30 ml 4% formaldehyde before being imaged on an
Applied Precision DeltaVision Deconvolution Microscope. Deconvolution andimage processing was performed using SoftWorx software.

ADP in HIV
FA-S Batiz et al
9

ACKNOWLEDGEMENTS
This work was supported by NHMRC program grant no. 510448. The views
expressed in this manuscript are those of the authors and do not reflect theviews of the US Army or the US Department of Defense. We thank the RV144
trial participants, the whole RV144 study team and all patients and blood
donors. We thank Dr Wendy Winnall for statistical advice.

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