Journal of Immunological Methods xxx (xxxx) xxx–xxx

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

Research paper

A multiplex assay for detection of SHIV plasma and mucosal IgG and IgA☆
Ivana K. Parkera, Krystin Ambrose Pricea, Tyana Singletaryb, Priya Srinivasana, James Smitha,
Kelly A. Curtisa,⁎
a
Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention,
Atlanta, GA, USA
b
Anyar Inc., Fort Walton Beach, FL, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Evaluating antibody maturation provides valuable data to characterize immune responses to HIV infection and
HIV antibodies can provide insight into biomedical intervention efficacy. It is important to develop assays that evaluate anti-
Simian immunodeficiency virus body maturation in both plasma and mucosal compartments. The nonhuman primate model provides a con-
Nonhuman primate trolled system to collect temporal data that are integral to assessing intervention strategies. We report the de-
Antibody affinity
velopment of a novel multiplex assay, based on the Bio-Plex platform, to evaluate plasma and mucosal IgG and
Mucous membrane
Immunoassay
IgA avidity and maturation against simian/human immunodeficiency virus (SHIV) in this controlled system.
Vaginal mucosa and plasma samples were collected from a prior study evaluating the efficacy study of a te-
nofovir disoproxil fumarate (TDF) intravaginal ring (IVR) against SHIVSF162P3 challenge in female pigtailed
macaques. For validation of the multiplex assay, specimens from six SHIV-infected placebo animals and one TDF
breakthrough animal were evaluated. For SHIV and HIV envelope analytes, antibody levels and avidity in both
compartments continued to mature post-infection. Maturation of IgG and IgA levels was similar in each com-
partment, however, mucosal antibody levels tended to be more variable. This SHIV assay elucidates IgG/IgA
antibody kinetics in the plasma and vaginal mucosa and will be a valuable tool in vaccine and other biomedical
intervention studies in the nonhuman primate model.

1. Introduction
Nonhuman primate (NHP) models of HIV infection have proven to
be instrumental for the advancement of human safety and efficacy trials
for pre-exposure prophylaxis (PrEP) and vaccine candidates
(McChesney and Miller, 2013; Anderson et al., 2016; Garcia-Lerma and
Heneine, 2012; Veazey, 2013). With the availability of new biomedical
interventions, including PrEP, it is important to understand the early
immune response dynamics of HIV infection, especially for individuals
who exhibit breakthrough infection, or become infected while receiving
antiretroviral treatment. Studies have shown that people who become
infected while receiving PrEP have an altered antibody response, which
may have implications for diagnosis using antibody-based tests (Curtis
et al., 2011; Laeyendecker et al., 2015). Furthermore, vaccine and
microbicide efficacy studies evaluating virus-specific antibody re-
sponses in the plasma and vaginal mucosa provide evidence of antibody
correlates of risk and protection, though the immunological interac-
tions are complex and depend upon many factors, including host
characteristics and treatment modalities (Tomaras and Plotkin, 2017;
Archary et al., 2016; Robinson, 2013; Voss et al., 2016). Simian/human
immunodeficiency virus (SHIV) infection of NHP demonstrates similar
antibody dynamics to those observed during HIV infection, and there-
fore presents a model that is useful for addressing important im-
munological questions (Evans and Silvestri, 2013; Hessell and
Haigwood, 2015; Pereira et al., 2012).
In the course of HIV infection, ongoing exposure to viral antigens
leads to an increased breadth of antibody specificity and avidity, which
continues to develop throughout the course of infection (Curtis et al.,
2011; Fiebig et al., 2003; Zetola and Pilcher, 2007; Suligoi et al., 2002).
HIV antibody has traditionally been detected using immunoassays such
as Western blot, enzyme-linked immunosorbent assays (ELISA), or en-
zyme immunoassays (EIA). In recent years, bead-based multiplex as-
says, based on the Luminex technology, have become increasingly at-
tractive for detection or quantification of antibody. The Bio-Plex
platform (Bio-Rad Laboratories, Inc., Hercules, CA) provides sensitive
detection of antibody responses with minimal sample volume, and has
the ability to detect up to 100 analytes in a single well. Luminex/Bio-
Plex assays have demonstrated improved performance compared to

☆
Disclaimer: The findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
⁎
Corresponding author at: Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, MS-A25, Atlanta, GA 30333, USA.
E-mail address: czv2@cdc.gov (K.A. Curtis).

http://dx.doi.org/10.1016/j.jim.2017.07.008
Received 1 May 2017; Received in revised form 13 July 2017; Accepted 24 July 2017
0022-1759/ © 2017 Published by Elsevier B.V.

Please cite this article as: Parker, I., Journal of Immunological Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.07.008
I.K. Parker et al.
ELISA and EIA due to the increased sensitivity and larger dynamic
range associated with the platform (Powell et al., 2013; Ayouba et al.,
2017; Satterly et al., 2017). Furthermore, the platform allows for flex-
ibility in terms of analyte evaluation, as Bio-Plex assays have been
developed to evaluate different antibody classes/subclasses, as well as
antibody avidity (Powell et al., 2013; Curtis et al., 2014; Curtis et al.,
2013a).
We have developed a SHIV-specific assay, based on the Bio-Plex
platform, which measures IgG and IgA antibody responses to SHIV in
plasma and vaginal mucosal eluates. To validate the performance of the
assay, antibody levels and avidity were evaluated in pigtailed macaques
(Macaca nemestrina) that were infected with SHIV as part of an in-
travaginal ring (IVR) PrEP efficacy study (Smith et al., 2015). Plasma
and vaginal sponge eluates were collected from a cohort of seven SHIV-
infected macaques (six controls and one PrEP breakthrough infection).
This SHIV-specific assay allows for the evaluation of longitudinal SHIV
IgG and IgA antibody responses in both the plasma and vaginal mucosa.
2. Materials and methods
2.1. Specimens
Previously established SHIV-negative (n = 46) and SHIV-positive
(n = 9) specimens were obtained from prior studies to establish assay
cut-off values for each analyte and to assess antibody specificity (Smith
et al., 2015; Smith et al., 2013; Ross et al., 2014; Pereira et al., 2015).
SHIV-SF162P3 is composed of the SIVmac239 backbone and a subtype
B HIV-1 envelope (Harouse et al., 2001). The archived specimens used
to validate the SHIV Bio-Plex assay have been described in detail
elsewhere (Smith et al., 2015). Briefly, TDF or placebo IVRs were in-
serted into twelve sexually mature depot medroxyprogesterone acetate
(DMPA)-treated female pigtailed macaques (n = 6 per group) and the
rings were replaced every four weeks. The macaques were exposed
vaginally once weekly for twelve weeks to SHIV162P3 (50 TCID50)
inoculations and the first exposure was administered one week after
IVR insertion. Macaques were monitored weekly for infection by RT-
PCR with a lower limit of detection of 50 copies/mL of plasma and
infection was confirmed by serology [Genetic Systems HIV-1/HIV-2
Plus O EIA (Bio-Rad Laboratories, Inc.)] (Smith et al., 2015). Virus
exposures were discontinued once SHIV RNA was detected in two
consecutive plasma specimens. The peak viral load of the breakthrough
animal was approximately 100-fold lower than the median viral load of
the placebo group. Vaginal secretions were collected monthly at each
IVR exchange using Ultra cell surgical sponges (3.5 × 4 mm; Beaver-
Visitec, Waltham, MA). Weekly collections ensued during a follow-up
period for all SHIV-infected animals (up to 4.5 weeks for the six placebo
IVR recipients, and 10 weeks for the one breakthrough TDF IVR animal)
and were subsequently used for this study.
2.2. Vaginal sponge elution
Vaginal sponges collected from pigtailed macaques were weighed
before and after collection of vaginal fluid to determine total fluid re-
covery. Vaginal fluid was eluted from the swabs as previously described
(Johnson et al., 2012). Briefly, the swabs were incubated for 30 min at
4 °C in 200 μl of elution buffer consisting of 1:100 dilution of a protease
inhibitor cocktail (P3480; Sigma-Aldrich, St. Louis, MO) in phosphate-
buffered saline (PBS). The eluate was separated from the swabs using
0.22 μm Costar Spin-X centrifuge tube filters (Sigma-Aldrich) by cen-
trifugation at 12,000 rpm for 20 min at 4 °C. The eluates were used
immediately for antibody testing.
2.3. SHIV Bio-Plex assay
A SHIV-specific multiplex panel was created by coupling the fol-
lowing recombinant proteins to COOH MagPlex Microspheres (Luminex
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Journal of Immunological Methods xxx (xxxx) xxx–xxx
Corporation, Austin, TX): SIV p27 Gag (mac239), HIV-1 envelope (Env)
gp70-V1V2 (Clade B/Case A2), HIV-1 Env gp140 and gp120
(SHIV162P3) (Immune Technology Corp, New York, NY). Additionally,
full-length recombinant subtype B HIV-1 Env proteins gp120 (IIIB),
gp160 (IIIB), and gp41 (MN) were obtained from ImmunoDX (Woburn,
MA). The coupling procedure was performed using a Bio-Plex Amine
coupling kit (Bio-Rad Laboratories), according to the manufacturer's
instructions. Briefly, 10 μg of recombinant protein was added to each
coupling reaction containing 1.25 × 106 microspheres. For sample
addition and non-specific binding controls, coupling reactions were also
performed using goat anti-human IgG (Invitrogen, Carlsbad, CA) or
goat anti-human IgA (SouthernBiotech, Birmingham, AL) and BSA
(Sigma-Aldrich), respectively.
Prior to testing, plasma samples were diluted 1:50 in PBS with 1%
BSA, 0.5% polyvinyl alcohol (PVA, Sigma-Aldrich), and 0.8% poly-
vinylpyrrolidone (PVP, Sigma-Aldrich); eluates from the vaginal swabs
were diluted 1:2. A working microsphere mixture was prepared by
combining 1 × 105 microspheres/ml of each microsphere set. The
MagPlex microspheres and pre-diluted samples were added in duplicate
to a 96-well plate (Millipore, Danvers, MA) at a volume of 50 μl each/
well and incubated for 30 min For measurement of antibody avidity,
the microspheres were washed once with assay buffer (PBS with 1%
BSA), re-suspended in 100 μl of assay buffer or 0.1 M diethylamine
(DEA), and incubated for 15 min. The microspheres were then washed
twice with assay buffer, re-suspended in 100 μl of assay buffer con-
taining 4 μg/ml phycoerythrin (PE)-labeled goat anti-human IgG
(Sigma-Aldrich), and incubated for 30 min. For detection of IgA anti-
bodies, PE conjugation to a goat anti-monkey IgA (Sigma-Aldrich) was
performed using an R-Phycoerythrin Conjugation Kit (Abcam,
Cambridge, MA), according to the kit instructions. The PE-labeled goat
anti-monkey IgA was added to the assay plate at 2.38 μg/ml in 100 μl of
assay buffer. The microspheres were washed, re-suspended in assay
buffer, and analyzed on a Bio-Plex 200 or 100 System (Bio-Rad
Laboratories). All incubations were performed on a plate shaker at
room temperature, with protection from light.
To control for run-to-run variation and to assess the coefficient of
variation (CV) for each analyte, a calibrator was included in every
plate. The calibrator was prepared by combining five HIV-1-positive
samples from commercially available panels (Zeptometrix Corp.,
Buffalo, NY), as described (Curtis et al., 2012). Calculated based on
reactivity to the calibrator, the CV for IgG was 26.3%, 12.6%, 14.9%,
and 14.8% for SHIV gp120, SHIV gp140, HIV-1 gp41, and SIV p27,
respectively. For IgA, the CV was 19.9%, 14.4%, 16.7%, and 27% for
SHIV gp120, SHIV gp140, HIV-1 gp41, and SIV p27, respectively. The
mean fluorescent intensity (MFI) values obtained by the Bio-Plex
System for plasma and vaginal samples were evaluated as a measure of
SHIV antibody levels. An avidity index (AI) was calculated for each
analyte using the formula: (MFI value of DEA-treated well / MFI value
of buffer-treated well) × 100. To calculate a cutoff value for each
analyte of the SHIV-specific multiplex assay that distinguishes ser-
opositive from seronegative, plasma samples from 46 SHIV-naïve
rhesus (M. mulatta) and pigtailed macaques were tested. Samples were
considered positive for an analyte if greater than two standard devia-
tions from the mean of the negative controls (Curtis et al., 2014; Curtis
et al., 2012; Curtis et al., 2013b). Seroconversion was defined as the
first time that a positive test result was identified by the Bio-Plex assay
for each individual analyte or by the HIV-1/HIV-2 Plus O EIA.
The nonparametric Wilcoxon rank-sum test was used to compare
SHIV Bio-Plex MFI values between SHIV-negative and -positive maca-
ques, as well as the time from viral RNA detection to seroconversion, as
measured by the EIA versus Bio-Plex analytes. Statistical analyses were
performed using the GraphPad Prism 6 Software (La Jolla, CA). A p
value of < 0.05 was considered significant.
I.K. Parker et al. Journal of Immunological Methods xxx (xxxx) xxx–xxx

Fig. 1. SHIV IgG and IgA antibody responses. Plasma specimens from known SHIV-negative and -positive rhesus macaques were tested with the SHIV multiplex assay for detection of IgG
(A) and IgA (B) antibodies. Median values (middle line) and standard deviations (top and bottom lines) for each sample set are denoted by solid lines. Statistically significant differences
(p ≤ 0.0001, Mann-Whitney test) are indicated by *, while lack of significance (p > 0.05) is indicated by ns.

3. Results
3.1. SHIV IgG and IgA antibody responses in plasma
The IgG antibody reactivities of SHIV-positive and -negative plasma
specimens against HIV-1 gp120, HIV-1 gp140, HIV-1 gp41, SIV p27,
SHIV gp120, SHIV gp140, and SHIV gp70 V1V2 are depicted in Fig. 1A.
A measurable distinction in MFI was seen between the SHIV-negative
and -positive specimens for all antigens (p ≤ 0.0001), with the excep-
tion of SHIV gp70 (p > 0.05). The positive control macaques included
in the evaluation did not exhibit plasma IgG reactivity to SHIV gp70.
The overall dynamic range in MFI values varied depending on the
specific antigen, with the greatest median MFI values observed for HIV-
1 gp41, SHIV gp120, and SHIV gp140. Similarly, a robust IgA antibody
response was observed with the SHIV-positive specimens relative to the
negative controls (Fig. 1B). As observed for IgG, no IgA reactivity to
SHIV gp70 was noted in the SHIV-positive macaques. The antigens that
exhibited the highest median MFI values for the SHIV-positive speci-
mens were HIV-1 gp41 and SHIV gp120. Overall, the dynamic range in
MFI values was smaller for IgA compared to IgG. Antibodies to gp70
were not detectable in the SHIV-positive specimens and, therefore,
gp70 could not be included in subsequent analyses.
3.2. Time to seroconversion
Plasma specimens from SHIV-positive pigtailed macaques, collected
as part of a PrEP efficacy study, were used to calculate the average time
from the first RNA-positive test result to seroconversion for each ana-
lyte tested (Fig. 2). The average time from the first RNA-positive result
to seroconversion, as measured by the HIV-1/HIV-2 Plus O EIA, was
2.2 weeks. For HIV-1 gp41, SHIV gp120, SHIV gp140 and SIV p27, the
average time to IgG seroconversion was 2, 2.2, 2, and 2.3 weeks,

3
I.K. Parker et al.
Fig. 2. Estimated time to seroconversion.
The average time from the first RNA-positive result to seroconversion, as measured by the
HIV-1/HIV-2 Plus O EIA and SHIV Bio-Plex assay, is summarized for each test/analyte.
Error bars represent one standard deviation from the mean.
respectively as measured by the Bio-Plex assay. For IgA, the average
time to seroconversion was 2, 2.3, 2.6, and 2.3 weeks for HIV-1 gp41,
SHIV gp120, SHIV gp140 and SIV p27, respectively. No statistical dif-
ference was observed between the average time for the EIA and any of
the Bio-Plex analytes (p > 0.05).
3.3. Longitudinal analysis of plasma IgG and IgA antibody responses
Longitudinal plasma IgG antibody responses from the SHIV-positive
animals are depicted in Fig. 3A. An overall increase in IgG antibody
levels and avidity was observed over time since the first RNA-positive
test result for SHIV gp120, SHIV gp140, HIV-1 gp41, and SIV p27. SHIV
gp120 and SHIV gp140 antibody levels continued to increase over time,
while HIV-1 gp41 IgG antibody levels increased, then plateaued two
weeks post RNA-positive result. In contrast to the envelope antigens,
the antibody response to p27 reached a plateau and had a sharp decline
at 12 weeks post-initial RNA-positive test result. The kinetics of the
plasma IgA antibody response was similar to the IgG response (Fig. 3B),
exhibiting an increase in antibody levels and avidity for SHIV gp120
and gp140 over time, as well as a rapid increase in HIV-1 gp41 and SIV
p27 antibody levels followed by a plateau and/or decline at around
12 weeks.
The PrEP breakthrough animal exhibited lower SHIV gp120 and
SHIV gp140 IgG and IgA antibody levels compared to the placebo group
(Fig. 3). The AI for these analytes was initially higher in the break-
through animal, but exhibited an overall blunted maturation curve as
compared to the placebo group. In contrast to the SHIV Env proteins,
SIV p27 IgG and IgA antibody levels and avidity continued to increase
over time in the breakthrough animal compared to controls.
3.4. Longitudinal analysis of mucosal IgG and IgA antibody responses
Longitudinal mucosal IgG and IgA antibody responses are displayed
in Fig. 4. IgG antibody levels increased over time from the first RNA-
positive test result for all analytes within specimens collected from the
vaginal mucosa. Mucosal IgA antibody levels also increased over time
for all analytes within the placebo treatment group (Fig. 4B). Similar to
the antibody reactivity observed with plasma, SIV p27 IgG and IgA
increased during the first 4 weeks post first RNA positive result. The
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Journal of Immunological Methods xxx (xxxx) xxx–xxx
peak in SIV p27 antibody levels was transient and levels declined to-
wards the latter time points of the study. For both IgG and IgA, antibody
avidity continued to increase for each analyte during the study follow-
up period. In general, the antibody responses exhibited greater varia-
tion between time points in the mucosal specimens, compared to
plasma, for both MFI values and avidity index.
In evaluating mucosal antibody responses in the PrEP breakthrough
animal, SHIV gp120, SHIV gp140, HIV-1 gp41, and SIV p27 IgG and IgA
antibody levels were lower in the breakthrough animal as compared to
the placebo group. Antibody avidity to SHIV gp120 and gp140 IgGs was
initially higher in the breakthrough animal, but showed no discernible
difference to the placebo group at approximately 8 weeks post first
RNA-positive result. Similar to plasma results, the breakthrough animal
mucosal avidity to SIV p27 IgG continued to mature throughout the
study and was higher when compared to controls. In contrast to mu-
cosal IgG, IgA avidity was blunted for SHIV gp120, gp140, and SIV p27
in the breakthrough animal. Although HIV-1 gp41 IgA antibody levels
were lower in the breakthrough animal compared to the placebo group,
there was no measureable distinction in avidity. However, these ob-
servations are from a single animal and, therefore, may not accurately
reflect the response in all breakthrough animals.
4. Discussion
We have developed an antibody-based multiplex assay for the de-
tection of SHIV IgG and IgA antibody responses to multiple antigens. To
evaluate the utility of the assay, we demonstrated detection of long-
itudinal antibody responses in both the plasma and vaginal mucosa
specimens from SHIV-positive pigtailed macaques. The SHIV NHP
model provides the opportunity to accurately assess longitudinal anti-
body responses, given that the timing of infection can be closely
monitored and specimen collection is carefully controlled. Dynamics of
the antibody response to SHIV provides key information regarding the
impact of partially protective PrEP and other biomedical interventions,
such as vaccines on the timing of seroconversion, antibody maturation
kinetics, and potential correlation with protection. Furthermore, de-
layed seroconversion or altered antibody maturation has important
implications for HIV diagnostics since the window period between in-
fection and detection may be increased for antibody-based diagnostic
tests (Keating et al., 2016). The customized SHIV antibody assay allows
for a direct comparison of the IgG/IgA antibody responses to be made
between the plasma and mucosal compartments. The SHIV multiplex
assay is a valuable tool for evaluating antibody responses using the NHP
model as a means to address crucial immunological questions per-
taining to HIV infection.
Previously, SHIV IgG antibody responses were evaluated in the
plasma of rhesus macaques that exhibited breakthrough infections
while receiving oral PrEP, using a customized SIV/HIV Bio-Plex assay
(Curtis et al., 2011). Oral PrEP treatment was associated with decreased
SHIV IgG antibody avidity in macaques that exhibited breakthrough
infection. Here, the SHIV multiplex assay was optimized to detect IgA,
as well as IgG, and included recombinant Env proteins derived from the
challenge virus (SHIV SF162P3) to improve recognition of the SHIV
antibodies to the assay antigens. As anticipated, the SHIV-positive
macaques exhibited a robust antibody response to SHIV gp120 and
gp140, with a larger dynamic range relative to HIV-1 (strain IIIB) de-
rived antigens that were used previously (Curtis et al., 2011). Given
that vaginal and rectal mucosa antibodies play an important role in the
delay of HIV replication and establishment of a productive infection,
the assay was also adapted to detect antibodies in vaginal mucosal
fluids collected from Wek Cel sponges (Smith et al., 2015). This is an
important tool to measure immune responses in studies evaluating ef-
ficacy of antiretrovirals or mucosal antibody distribution following
vaccination or immunoprophylaxis. Although not addressed in the
current study, the sample preparation step for the customized SHIV
multiplex assay can also be optimized to measure antibody responses in
I.K. Parker et al.
Fig. 3. Longitudinal SHIV IgG and IgA antibody responses in plasma.
SHIV plasma IgG (A) and IgA (B) antibody levels and avidity, as measured by the customized SHIV Bio-Plex assay, were plotted over weeks since first detectable RNA test. The median
mean fluorescent intensity (MFI) and avidity index (AI) values for the placebo group (n = 6) are represented by solid lines and the PrEP breakthrough animal (n = 1) is represented by a
dotted line.
the rectal compartment (Powell et al., 2013).
A 2015 study by Smith et al. demonstrated that an IVR containing
TDF provided protection in DMPA-treated pigtailed macaques against
vaginal SHIV challenge (Smith et al., 2015). Here, we compared the
appearance of SHIV IgG and IgA antibodies in the placebo, as measured
by the customized SHIV multiplex assay, relative to the detection of
viral RNA. In humans, the estimated median time between detection of
HIV-1 RNA, as measured by the APTIMA HIV-1 RNA Qualitative Assay
(Gen-Probe, Inc., San Diego, CA), and the HIV-1/HIV-2 Plus O EIA is
about 14 days (Delaney et al., 2017; Masciotra et al., 2015). A similar
window between the first SHIV RNA-positive test result and ser-
oconversion, as measured by the EIA and SHIV multiplex assay, was
observed (approximately 2–2.5 weeks), which further strengthens the
translational value of this multiplex assay in the SHIV model for HIV
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Journal of Immunological Methods xxx (xxxx) xxx–xxx
research. Notably, there was no statistical difference between the time
to detection for the EIA and all SHIV Bio-Plex analytes, given that the
HIV-1/HIV-2 Plus O EIA detects both IgG and IgM antibodies, which
reduces the window of detection relative to tests that only detect IgG
antibodies (Delaney et al., 2017). It is likely that the similar time to
detection between the EIA and SHIV multiplex assay was reflective of
the sensitivity of the Bio-Plex format.
In addition to the timing of seroconversion, SHIV antibody kinetics
in the placebo group were similar to the longitudinal antibody re-
sponses observed in the plasma of HIV-1-infected humans (Curtis et al.,
2012; Curtis et al., 2013b). We previously demonstrated that HIV-1 Env
antibodies and antibody avidity continue to increase post-infection in
humans; therefore, it was encouraging to observe a similar robust an-
tibody response to SHIV Env antigens in SHIV-infected pigtailed
I.K. Parker et al.
Fig. 4. Longitudinal SHIV IgG and IgA antibody responses in the vaginal mucosa.
SHIV vaginal mucosa IgG (A) and IgA (B) antibody levels and avidity, as measured by the customized SHIV Bio-Plex assay, were plotted over weeks since 1st detectable RNA test. The
median MFI and AI values for the placebo group (n = 6) is represented by a solid line and the PrEP breakthrough animal (n = 1) is represented by the dotted line. Note the difference in
scale for the AI of the vaginal mucosa specimens relative to the plasma.
macaques. The decline in SIV p27 antibody levels noted at later time
points is consistent with a decline or stabilization of virus replication
and/or formation of antigen-antibody immune complexes (Smith et al.,
2015; Henrard et al., 1995). Studies have shown that HIV IgG levels
typically predominate over IgA in the vaginal mucosa (Archary et al.,
2016; Raux et al., 1999). Although the SHIV multiplex assay does not
fully quantitate antibody levels (semi-quantitative), the MFI scale for
SHIV IgG versus IgA indicated a greater IgG response relative to IgA in
both the plasma and vaginal mucosa. Similar to previous reports in HIV-
1-infection, we noted comparable antibody maturation curves between
the plasma and vaginal mucosa, which may reflect some transudation of
systemic antibody to the genital tract, as described previously (Archary
et al., 2016; Raux et al., 1999). Although the overall trend in long-
itudinal antibody responses was similar in the plasma and vaginal
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Journal of Immunological Methods xxx (xxxx) xxx–xxx
mucosa, the antibody levels and avidity tended to be more variable
between time points in the mucosal specimens. It is not known whether
the erratic antibody responses are an artifact of the sampling method or
inconsistent collection of antibodies from specimen to specimen. SHIV-
specific antibody levels were normalized by total IgG or IgA (data not
shown); however, normalization did not improve the variability of the
mucosal antibody responses. Further investigation is needed to de-
termine the potential cause of the variability.
Although a single breakthrough infection does not provide sufficient
statistical power to examine differences between the placebo group and
PrEP breakthrough infection, the breakthrough animal was included in
the assay evaluation for a qualitative comparison. The breakthrough
animal continued to have the TDF IVR during the entire study follow-up
period, which resulted in lower plasma viremia than the placebo group,
I.K. Parker et al.
so it is not an unexpected finding that antibody levels and avidity were
lower for most analytes compared to the placebo group. Initiation of
ART is recommended upon HIV diagnosis; therefore, an individual that
becomes infected while receiving PrEP may exhibit a similar reduction
in antibody levels with the continued presence of ARV's. One interesting
finding in the breakthrough animal was a transient elevated antibody
avidity to gp120 and gp140, relative to the placebo group, within the
first six weeks of detectable infection. Given that the breakthrough
animal was infected after a significantly greater number of exposures
(8) compared to the placebo group (n = 6, median of 2: 1,2, 2, 2, 4 and
8 exposures, respectively) (Smith et al., 2015), the initial elevation in
antibody avidity may be a result of multiple exposures to the Env an-
tigens prior to a productive SHIV infection. Another unique finding was
the elevated SIV p27 antibody levels and avidity in plasma of the
breakthrough animal compared to the placebo group. A similar increase
in p24 antibody response was observed in HIV-1-infected individuals
that became infected in the presence of a topical tenofovir gel (Archary
et al., 2016). Since only a single breakthrough infection was included in
this study, it cannot be concluded whether the antibody kinetics ob-
served for this animal will be typical of all breakthrough infections.
Breakthrough animals are rare in PrEP studies and the multiplex assay
may offer insight into why these particular animals became infected.
In our study, specimens from a prior PrEP efficacy study were used
to validate performance of the SHIV multiplex assay. A limited number
of SHIV-negative and SHIV-positive specimens were available for the
assay optimization; therefore, follow-up studies are needed to further
explore the performance characteristics of the assay (sensitivity, spe-
cificity, etc.). In order to examine the effects of PrEP or other treatment
strategies on the antibody response to SHIV/HIV, a larger number of
study participants are needed to make an effective comparison. For the
purpose of the current study, median avidity values were plotted to
facilitate the visualization of the antibody kinetics, however, variation
in antibody reactivity for each individual or animal should be con-
sidered to discern immune differences between treatment versus pla-
cebo groups. Given the versatility of the Bio-Plex platform, the SHIV
multiplex assay can be further optimized to detect both SIV and SHIV
antigens in different species of macaques, expand upon the analyte
panel to cover other sexually transmitted pathogens that are commonly
associated with HIV infection, and detect additional antibody classes/
subclasses. Furthermore, the SHIV assay may be used to explore the
relationship between SHIV antibody maturation and viral reservoir size
in nonhuman primate studies evaluating curative interventions.
In summary, we have demonstrated that the SHIV multiplex assay is
a useful tool for addressing immunological questions that may be ap-
plied towards the advancement of HIV treatment and intervention
methods or to inform HIV diagnostics in the age of PrEP. In the NHP
model, we have the capability to fully investigate the immunological
contribution in decreased viral peak and set point that have been ob-
served in PrEP failures and apply this knowledge to clinical outcomes
(Smith et al., 2015; Garcia-Lerma et al., 2008; Cong et al., 2016).
Acknowledgments
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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