Design and Analysis of Rhesus Cytomegalovirus IL-10
Mutants as a Model for Novel Vaccines against Human
Naomi J. Logsdon1, Meghan K. Eberhardt2, Christopher E. Allen1, Peter A. Barry2, Mark R. Walter1*
1Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 2Center for Comparative Medicine, University of
California Davis, Davis, California, United States of America
Background: Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-
10). Despite only ,26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-
10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low
sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that
specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with
functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.
Methods and Findings: Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon
vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations
were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate
RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-
type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.
Conclusion: This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-
functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic
strategies, to nullify HCMV’s ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary
mucosal infection, and (3) establish a lifelong persistent infection.
Citation: Logsdon NJ, Eberhardt MK, Allen CE, Barry PA, Walter MR (2011) Design and Analysis of Rhesus Cytomegalovirus IL-10 Mutants as a Model for Novel
Vaccines against Human Cytomegalovirus. PLoS ONE 6(11): e28127. doi:10.1371/journal.pone.0028127
Editor: Chandra Verma, Bioinformatics Institute, Singapore
Received July 26, 2011; Accepted November 1, 2011; Published November 21, 2011
Copyright: ? 2011 Logsdon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants R01 AI49342 (to PAB) and RO1 AI047300 (to MRW) from the National Institute of Allergy and Infectious Diseases
(www.niaid.nih.gov) of the National Institutes of Health (NIH), and by a grant RR00169 from the National Center for Research Resources (NIH) to the California
National Primate Research Center (www.cnprc.ucdavis.edu). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Human cytomegalovirus (HCMV) is a ubiquitous human b-
herpesvirus (50-.95% adult seroprevalence worldwide) that can
infect a susceptible individual at any time during pre- or post-natal
life . HCMV infection is generally subclinical in those with
functional immune systems. However, HCMV establishes and
maintains a lifelong persistence despite a robust host immune
response. In fact, ,10% of memory CD4+and CD8+T-cells in
long-term infected hosts are HCMV-specific , and generate
antibodies against multiple HCMV glycoproteins that neutralize
the virus [3,4]. Persistence is characterized by the presence of cells
harboring essentially quiescent HCMV genomes that can
asymptomatically reactivate to produce infectious virions that
can be shed in bodily fluids, such as breast milk, saliva, and urine.
Serious HCMV-induced clinical outcomes can occur in those
with immature or compromised immune systems, including
congenitally infected newborns, immunosuppressed transplant
recipients, and immunodeficient AIDS patients . Transplacen-
tal transmission from mother to fetus can occur during primary
HCMV infection of the mother, reactivation of persistent virus
within the mother, or maternal re-infection. In the case of
maternal re-infection, the demonstration that 10% of seropositive
women who give birth to a congenitally infected infant acquired
new antigenic reactivity to HCMV antigens between pregnancies
is indisputable evidence that prior immunity is incompletely
protective against reinfection with antigenic HCMV variants .
These results further suggest that reinfection with HCMV leads to
attenuation of antiviral effector/memory functions, enabling
progeny virions to ultimately disseminate beyond the mucosal site
of reinfection to the maternal/fetal interface. In both solid organ
(SOT) and bone marrow transplantation (BMT), resident HCMV
genomes can reactivate under conditions of iatrogenic immuno-
suppression. For HIV-infected individuals, resident HCMV
genomes can reactivate during onset of immunodeficiency and
cause end-organ disease, such as retinitis .
Since HCMV was recognized as an infectious threat to the
fetus, there have been repeated calls for a vaccine that prevented
PLoS ONE | www.plosone.org1November 2011 | Volume 6 | Issue 11 | e28127
congenital infection in women without preconceptional immunity
to HCMV [8,9,10,11]. The advent of solid organ and bone
marrow transplantation as medical options has heightened the
need for an HCMV vaccine to protect immunosuppressed
recipients from fulminant HCMV infections. Progress on a
vaccine has been made using glycoprotein B (gB) in clinical trials
designed to protect seronegative women with children from
primary infection, and seronegative transplant recipients from
HCMV infection and/or disease post allograft [12,13]. Both trials
achieved measurable (,50%) successes in reducing the rate of
acquisition of HCMV, the extent of HCMV replication and length
of anti-HCMV drug therapies, respectively. The absence of
complete protection in both trials argues that further vaccine
optimization is required to eliminate the risk of pathogenic
outcomes associated with HCMV infection, re-infection, and/or
reactivation. One reason for sub-optimal performance of the
current gB-vaccines may be the absence of other, viral proteins
that could increase vaccine-mediated protective efficacy. One such
class of viral proteins that has not been investigated consists of the
HCMV-encoded immuno-modulatory proteins that are thought to
be critical viral elements responsible for attenuation of host
immunity in vivo .
The HCMV IL-10 ortholog, CMVIL-10, is one such viral
immune modulating protein that offers several potential advan-
tages for vaccination. Despite only 26% amino acid sequence
identity between CMVIL-10 and cellular IL-10 (cIL-10), CMVIL-
10 retains the immunosuppressive properties of cIL-10 on multiple
lymphoid cell types, especially dendritic cells (DC), which link
innate and adaptive immunity [15,16,17,18,19]. In addition, cIL-
10 and CMVIL-10 both engage the IL-10R1 and IL-10R2 cell
surface receptor chains to induce their biological activities
[19,20,21,22]. Binding studies demonstrate cIL-10 and CMVIL-
10 form similar high affinity (,1 nM) interactions with the IL-
10R1 chain and low affinity (,mM) contacts with the IL-10R2
chain [22,23]. As a result, the IL-10/IL-10R1 interaction occurs
first, followed by the assembly of the IL-10/IL-10R1/IL-10R2
ternary complex, which activates intracellular kinases (Jak1 and
Tyk2) and transcription factors (STAT3) leading to IL-10 cellular
responses . The importance of viral IL-10 in vivo is highlighted
by a recent study showing that primary infection of rhesus (Rh)
macaques with a variant of RhCMV lacking the RhCMVIL-10
gene led to (1) increased innate responses at the site of inoculation,
and (2) increased long-term B and T cell responses to RhCMV
antigens, compared to infection with the parental variant
expressing RhCMVIL-10 . These studies suggest a mechanism
by which early interactions between viral IL-10 and DC at the site
of infection skew the adaptive responses to a state favoring viral
persistence. The precise role of CMVIL-10, relative to cIL-10,
remains to be determined. However, studies show CMVIL-10
induces cIL-10 in DC and trophoblasts and prevents effective T-
cell priming by inhibiting dendritic cell (DC) maturation and
priming [16,24]. Furthermore, the CMVIL-10 open reading
frame (ORF) is conserved in numerous culture-adapted strains and
clinical isolates of HCMV . Together, these data suggest
CMVIL-10 is an attractive target for vaccine development.
To test this hypothesis in an animal model, we sought to use
RhCMVIL-10 as an antigen for vaccination studies in rhesus
macaques (Macacca mulatta). The use of the RhCMV model was
essential since, in contrast to murine CMV, RhCMV encodes a
viral IL-10 (RhCMVIL-10) and displays similar infection routes,
seroconversion rates, and sheds virus in urine and saliva as
observed for HCMV . However, since RhCMVIL-10 exhibits
potent immunosuppressive activities that disrupt immune function,
wild-type (WT) RhCMVIL-10 was not appropriate for our studies.
To disrupt RhCMVIL-10 biological activity, we designed two
RhCMVIL-10 mutants, guided by crystal structures of the cIL-
10/IL-10R1 and CMVIL-10/IL-10R1 complexes that could not
bind IL-10R1 [21,27]. The designed mutants fail to induce
RhCMVIL-10 biological activities. Vaccination of RhCMVIL-10-
infected rhesus macaques with the inactive RhCMVIL-10 mutants
generated anti-RhCMVIL-10 Abs that block the biological activity
of wild-type RhCMVIL-10 and do not cross react with cellular
Expression, purification, and characterization of
RhCMVIL-10 was expressed in Drosophila Schneider S2 cells
and purified by affinity chromatography using agarose beads
coupled with the human IL-10R1 chain as previously described
. Affinity purified RhCMVIL-10 ran as two bands on SDS-
PAGE gels. The major band exhibited a molecular weight (MW)
of ,19 kilodaltons (kDa), while a much less intense second band of
,18 kDa was also observed (Fig 1). The presence of two bands
suggested RhCMVIL-10, expressed in insect cells, is predomi-
nantly glycosylated on its single N-linked glycosylation site, Asn-87
(Fig 2A). Affinity purified RhCMVIL-10 fractionated from a size
exclusion column at essentially the same position as cIL-10, which
was previously shown to form a noncovalent homodimer
[28,29,30]. The combined results of the SDS-PAGE and GF data
are most consistent with the interpretation that RhCMVIL-10 is a
glycosylated non-covalent homodimer.
Design of RhCMVIL-10 point mutants defective in IL-10R1
Crystal structures of the cIL-10/IL-10R1 and CMVIL-10/IL-
10R1 complexes, combined with sequence analysis, were used to
assist in the design of RhCMVIL-10 point mutants that could not
bind to the IL-10R1 chain (Fig 2). Mature cIL-10 and RhIL-10
sequences share 95% sequence identity, while the viral IL-10s
Figure 1. Purification and quaternary structure of RhCMVIL-10.
GF Chromatographs of cIL-10 (grey) and RhCMVIL-10 (black) plotted
with X-axis in mL and Y-axis in optical density (OD) at 280 nm. (Inset)
SDS-PAGE gel of affinity purified RhCMVIL-10 (lane 1) and pooled
fractions (black bar) of the major RhCMVIL-10 GF peak (lane 2).
Design and Analysis of RhCMVIL-10 Mutants
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(RhCMVIL-10 and CMVIL-10) exhibit between 23%-28%
identity with each other and with the cellular IL-10s. Despite
the divergent amino acid sequences, five surface exposed residues
(helix A residues Arg-27, Gln-38; and helix F residues Glu-142,
Asp-144, and Glu-151) are conserved in all four sequences and
form extensive contacts with IL-10R1 in the cIL-10/IL-10R1 and
CMVIL-10/IL-10R1 binding interfaces (Fig. 2) [21,27]. The
conserved interactions made by these five residues suggested the
crystal structures of the cIL-10/IL-10R1 and CMVIL-10/IL-
10R1 provide suitable structural models for selecting mutations
that disrupt RhCMVIL-10/RhIL-10R1 interactions. The struc-
tural data are critical, since the goal is to design biologically
inactive RhCMVIL-10 mutants that preserve the wild-type
RhCMVIL-10 three-dimensional structure.
Using the structural information described above (Fig 2B),
mutations of three RhCMVIL-10 residues (Gln-38, Glu-142, and
Asp-144) were predicted to maximally disrupt RhCMVIL-10/
RhIL-10R1 interactions and prevent RhCMVIL-10 biological
activity. These residues were chosen based on the following
criteria. 1) The residues were conserved among the human and
viral IL-10 amino acid sequences, including CMVIL-10 from
different HCMV strains; 2) the amino acids made extensive
contacts with IL-10R1 in the cIL-10/IL-10R1 and CMVIL-10/
IL-10R1 complexes; and 3) the residues were located in the center
of the IL-10/IL-10R1 interfaces. This final criterion was required
because RhCMVIL-10 mutations predicted to disrupt IL-10R1
binding, but located on edge of the IL-10/IL-10R1 interface,
might assume altered side-chain conformations that could still
enable efficient IL-10R1 binding. To test this latter hypothesis, an
Arg-34Glu RhCMVIL-10 mutant was made. The corresponding
amino acid in cIL-10 and CMVIL-10 is Lys-34, which forms salt-
bridge interactions with IL-10R1 residues Asp-100 and Glu-101
on the edge of the IL-10/IL-10R1 interface (Fig 2B). As described
for Arg-34Glu, amino acid changes at the identified positions were
chosen to sterically prevent RhCMVIL-10/IL-10R1 complex
formation and/or disrupt extensive hydrogen bonding networks
found in the interface.
Characterization of RhCMVIL-10 mutant binding to
Based on the analysis above, five RhCMVIL-10 point mutants
(Gln-38Arg, Gln-38Tyr, Arg-34Glu, Glu-142Tyr, Asp-144His)
were expressed in insect cells, and the supernatants tested for
binding to the IL-10R1 chain (Fig 3). To assist in purification, and
provide a common epitope for detecting the RhCMVIL-10
mutants, a 6-residue histidine tag (H6) and a factor Xa protease
site (Fxa) was added to the N-terminus of these RhCMVIL-10
mutants (H6FXa-RhCMVIL-10). Western blot analysis revealed
H6FXa-RhCMVIL-10 mutants Gln-38Tyr, or Glu-142Tyr, were
not efficiently expressed (Fig. 3 bottom). The other 3 RhCMVIL-
10 mutants (Arg-34Glu, Gln-38Arg, and Asp-144His) were
expressed at comparable levels (Fig. 3 bottom), but were unable
to bind to IL-10R1 coupled beads (Fig. 3 top). In contrast, WT
RhCMVIL-10 was able to bind to IL-10R1 beads.
Figure 2. Sequence and structure model of RhCMVIL-10 binding residues. (A) Sequence alignment of cellular and viral human and rhesus
IL-10s. The predicted site for N-linked glycosylation in RhCMVIL-10 is underlined. GenBank Accession numbers: RhCMVIL-10, AAF59907; cIL-10,
AAA63207; RhIL-10, AAA99975; CMVIL-10, AAF63437. CIL-10 helices are denoted on the alignment (A–F ), which also highlights residues chosen
for mutagenesis. (B) Structure model of the RhCMVIL-10/RhIL-101R1 interface based on the crystal structure of the HuIL10/HuIL-10R1 complex (pdbid
1Y6K,  ). Residues chosen for mutagenesis that disrupt IL-10R1 binding are shown in red, while the Arg-34Glu mutant (corresponding to Lys-34 in
CMVIL-10) that exhibits essentially WT activity is colored green (see Fig. 4A).
Figure 3. Expression and IL-10R1 binding of RhCMVIL-10 point
mutants. Expression of RhCMVIL-10 point mutants in Drosophila cell
media was characterized by western-blotting (WB). Cell supernatants
containing the point mutants were incubated with HuIL-10R1 coupled
beads. After washing, the beads were loaded onto a 12% SDS-PAGE gel
and subsequently stained with coomassie blue.
Design and Analysis of RhCMVIL-10 Mutants
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Analysis of RhCMVIL-10 mutants ability to proliferate
human TF1/IL-10R1 cells
To characterize the RhCMVIL-10 point mutants (Gln-38Arg,
Arg-34Glu, and Asp-144His) in a more sensitive assay, we tested
the ability of each mutant to stimulate proliferation of TF-1/IL-
10R1 cells. TF-1/IL-10R1 cells are a human erythroleukemic cell
line stably transfected with the human IL-10R1 chain, which
causes them to proliferate upon addition of IL-10 to the media
. The concentrations of RhCMVIL-10, and RhCMVIL-10
point mutants, in Drosophila cell supernatants were estimated by
SDS-PAGE. Serial dilutions of each RhCMVIL-10 mutant and
cIL-10 were added to TF-1/IL-10R1 cells and proliferation was
measured after 2 days. As shown in Figure 4, RhCMVIL-10 and
cIL-10 exhibited essentially equivalent biological activity in the
assay. RhCMVIL-10 Arg-34Glu also exhibited essentially wild-
type biological activity, presumably because of its location on the
edge of the IL-10/IL-10R1 interface (Fig 2). In contrast,
RhCMVIL-10 Gln-38Arg and RhCMVIL-10 Asp-144His exhib-
ited effective concentrations (EC50s) that were ,100- and ,300-
fold lower than RhCMVIL-10 and cIL-10 (Fig. 4), but still
retained considerable activity at the highest concentration tested
Since the single mutants retained some activity, we tested two
RhCMVIL-10 double mutations in the TF-1/IL-10R1 cell assay
(Fig 4B). RhCMVIL-10 mutant 1 (M1) consisted of Gln-38Arg
and Asp-144His point mutations, while RhCMVIL-10 mutant 2
(M2) contained Glu-142Gln and Asp-144His mutations. Because
of the poor solubility of the H6Fxa-RhCMVIL-10 proteins during
initial purification studies, a C-terminal Fxa and H6 tag
(RhCMVIL-10-FXaH6) was added to RhCMVIL-10 M1 and
M2. Serial dilutions of M1 and M2, based on protein
concentrations estimated from SDS-PAGE gels, were added to
TF-1/IL-10R1 cells, which revealed RhCMVIL-10M1 and M2
did not exhibit biological activity at concentrations as high as
750 ng/mL (Fig. 4B).
Analysis of RhCMVIL-10 mutants ability to suppress IL-12
from Rhesus PBMC
Based on the results of the TF-1/IL-10R1 assays, RhCMVIL-
10 double mutants, M1 and M2, were purified by nickel affinity
chromatography. Purified M1 and M2 proteins were assayed for
their ability to inhibit the production of IL-12 in rhesus PMBCs
stimulated with lipopolysaccaride (LPS) (Fig. 5). Addition of LPS
to PBMC from three different animals stimulated high levels of IL-
Figure 4. Biological activity of RhCMVIL-10 mutants M1 and M2 on TF1/IL-10R1 cells. (A) Cell supernatants containing RhCMVIL-10 single
point mutants (described in the Figure legend) (Lys-34Glu, Gln-38Arg, and Asp-144His) were evaluated for their ability to stimulate proliferation of TF-
1/IL-10R1 cells in relation to cIL-10 and WT RhCMVIL-10. (B) TF-1/IL-10R1 cell proliferation assay for RhCMVIL-10 M1 (Q38R, D144H) and M2 (E142Q,
D144H) cell supernatants. Error bars represent estimated standard error from duplicate measurements.
Design and Analysis of RhCMVIL-10 Mutants
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12 expression, relative to PBMCs incubated in media alone. In
marked contrast, co-incubation of LPS-activated PBMC with WT
RhCMVIL-10 strongly inhibited IL-12 expression to levels below
those observed in un-stimulated cells. In contrast to WT
RhCMVIL-10, RhCMVIL-10 M1 and M2 could not inhibit IL-
12 production in LPS treated PBMC cultures at any concentration
tested (0.1 ng/mL-1000 ng/mL). Thus, RhCMVIL-10 M1 and
M2 were essentially devoid of functional activity (,88-100%
inactive, Fig. 5), as previously shown in the TF-1/IL-10R1 cell
proliferation assay (Fig. 4).
Characterization of RhCMVIL-10/IL-10R1 interactions by
surface plasmon resonance (SPR)
Surface plasmon resonance (SPR) experiments were performed
to further validate that RhcmvIL-10 M1 and M2 exhibit reduced
binding to IL-10R1. The RhCMVIL-10 dimer (0–100 nM), or
RhCMVIL-10 mutants (100 nM, 500 nM, and 1000 nM), were
injected over a Biacore chip surface of human IL-10R1, formed by
attaching an IL-10R1-FC fusion protein to a CM5 chip using an
anti-FC antibody (Fig 6). Human and rhesus IL-10R1 share 94%
amino sequence identity, suggesting human IL-10R1 can provide
a reasonable estimate of RhCMVIL-10/RhIL-10R1 interactions.
WT RhCMVIL-10/IL-10R1 sensorgrams were globally fit to a
bivalent analyte kinetic model (Fig 6). The resulting binding
constants reveal an initial interaction between RhCMVIL-10 and
human IL-10R1 of 65.1 nM (KD1), with a second apparent
binding constant, KD2, of 1.3 nM (Table 1). Interpretation of the
binding constants in this kinetic model is difficult. However, they
provide a baseline for comparing RhCMVIL-10 M1 and M2
binding to IL-10R1.
RhCMVIL-10 M1 and M2 were injected at three concentra-
tions (100 nM, 500 nM, and 1000 nM) over the IL-10R1-FC
surface (Fig 6). For RhCMVIL-10 M1, the maximal response unit
(RU) obtained at 1000 nM was 0.8 RU, which is 3.8% of the
maximal RU obtained for RhCMVIL210 at 100 nM (20.9 RU).
KD1 and KD2 values for RhCMVIL-10M1 were estimated to be
43 mM and 410 nM, respectively (Table 1). RhCMVIL-10 M2
bound slightly better to IL-10R1 than RhCMVIL-10 M1. A
maximal response of 3 RU was obtained from the 1000 nM
injection of RhCMVIL-10 M2. KD1 and KD2 values for
RhCMVIL-10 M2 were 14.7 mM and 94.4 nM, respectively
(Table 1). Comparing the KD1 parameters suggests RhCMVIL-
10 M1 and M2 exhibit at least ,226–660 fold lower affinity for
IL-10R1. However, this estimate is likely low since the impact of
disrupting the second interaction in the dimer has not been
considered. Thus, consistent with the bio-activity data (Figs 4 and
5), the SPR studies demonstrate the ability of RhCMVIL-10 M1
and M2 to bind IL-10R1 has been extensively disrupted, relative
to wild-type RhCMVIL-10.
Immunization of rhesus macaques with RhCMVIL-10M1
Based on the results of the SPR studies and the cell-based assays,
RhCMVIL-10 M1 and M2 were used to immunize six RhCMV-
infected rhesus macaques (Table 2) to determine whether
functionally inactivated RhCMVIL-10s could generate antibodies
that neutralize WT RhCMVIL-10 biological activity (RhCMVIL-
10-NAbs). Immunization of RhCMV- infected animals was
performed since disruption of the IL-10 signaling pathway has
been proposed as a therapeutic intervention to alter the course of
persistent pathogen infections (see Discussion). Thus, using
infected animals, allowed us to survey their pre-vaccination
RhCMVIL-10-NAb status and determine if vaccination with M1
or M2 could increase RhCMVIL-10-NAb levels. The production
of RhCMVIL-10-NAbs during the immunization procedure was
monitored by testing the ability of plasma, collected throughout
the immunization schedule (Table 2), to antagonize RhCMVIL-
10-mediated inhibition of IL-12 in LPS stimulated PBMC (Fig 7).
Using this assay, the presence of RhCMVIL-10-NAbs was
evaluated at the time of the initial DNA vaccination (Vx) (week
0), after the third DNA booster immunization (week 8), and six
times following the two protein boosts at weeks 14 and 18 (Fig 7).
Figure 5. Ability of RhCMVIL-10 M1 and M2 to suppress IL-12 levels in LPS-activated rhesus PBMC. IL-12 levels produced by LPS-
activated rhesus PBMCs were measured by ELISA in the presence, or absence, of purified RhCMVIL-10 M1, RhCMVIL-10 M2, or RhCMVIL-10 WT over a
concentration range of 0.1–1,000 ng/ml. Also shown are results for cells incubated with media alone and with LPS alone. Assays were performed
using PBMC from three rhesus macaques (Animal 1–3).
Design and Analysis of RhCMVIL-10 Mutants
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Figure 6. SPR analysis of RhCMVIL-10 wild-type, M1, and M2 binding to IL-10R1. Panels A, B, and C, show experimental sensorgrams
(colored) and bivalent model fits (black lines) for RhCMVIL-10WT, M1, and M2, respectively. Note the Y axis in panels B and C only extends to +/- 5RU
compared to 25 to 25RU in panel A. The maximum RU observed for RhCMVIL-10 M1 and M2 is below that of RhCMVIL-10WT observed at 3.125 nM;
the lowest concentration tested.
Design and Analysis of RhCMVIL-10 Mutants
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Immunized animals did not display any detectable changes in
activity, eating, or grooming, suggesting that the immunization
was well tolerated. Three of the six immunized animals exhibited
demonstrable RhCMVIL-10-NAb titers at the time of the first
DNA immunization, ranging from 5 – 80% neutralization of
RhCMVIL-10 biological activity (week 0) (Fig. 7). All six animals
exhibited RhCMVIL-10 binding antibodies (BAb) at the time of
the first DNA vaccination (Fig 7A). DNA immunization at weeks
0, 4, and 7 did not stimulate increases in RhCMVIL-10-NAb
responses. However, RhCMVIL-10-NAb titers were prominently
increased in five of the six vaccinees after the first and/or second
protein vaccinations at weeks 14 and 18 (Fig 7). One vaccinee with
undetectable RhCMVIL-10-NAb responses prior to immunization
did not develop detectable RhCMVIL-10-NAb responses after
either protein boost (Fig 7B, open circles). However, increases in
BAbs, following the two protein boosts, were observed for this
animal (Fig 7A, open circles). The animal that failed to respond
was vaccinated with the M2 protein. While M2 might be
considered a poorer immunogen than M1, based on our pre-
vaccination survey (week 0), some naturally infected animals fail to
develop RhCMVIL-10-NAbs, despite the presence of RhCMVIL-
10-binding antibodies (Fig 7).
Plasma from animals immunized with M1 and M2 does
not cross react with cellular RhIL-10
RhCMVIL-10 shares only 23% amino acid sequence identity
with RhIL-10, and there are no more that 3 contiguous amino
acids that are identical when the two proteins are aligned. This
suggested that RhCMVIL-10-NAbs generated by immunization
with RhCMVIL-10 M1 or M2 would not cross react with cellular
RhIL-10. To confirm this hypothesis, plasma samples from each
animal in Figure 7, were tested for the ability to neutralize the
biological activity of cellular RhIL-10 activity. This was performed
by determining whether plasma samples from immunized animals
that neutralized RhCMVIL-10, similarly neutralized the ability of
RhIL-10 to suppress IL-12 expression in LPS-activated rhesus
PBMC. As shown in Figure 8, week 19 plasma samples (Fig 7) that
completely neutralized RhCMVIL-10-mediated suppression of IL-
12 had no effect on RhIL-10-induced suppression of IL-12.
In this study, structural biology was used to rationally design
non-functional RhCMVIL-10 immunogens for vaccination in a
nonhuman primate model that closely mimics HCMV infection in
humans. Using this approach, we have established that immuniz-
ing animals with signaling-incompetent RhCMVIL-10 mutants
(M1 and M2) is able to generate, or boost, RhCMVIL-10-NAb
titers in seropositive rhesus macaques. To our knowledge, this is
the first report of a rational vaccine/therapeutic strategy to disrupt
a viral cytokine, CMVIL-10.
Blockade of CMVIL-10 signaling is supported by numerous
studies that demonstrate evolutionarily distinct mammalian
pathogens exploit the IL-10/IL-10R1 signaling pathway to subvert
protective immunity. Multiple viruses (e.g. CMV [15,32], Lym-
phocytic Choriomeningitis Virus – LCMV [33,34], Dengue
[35,36], HIV [37,38], human papillomavirus , Hepatitis B
Table 1. SPR Binding Constants for RhCMVIL-10 / human IL-10R1 Interactions.
ka1 (1/Msec)kd1 (1/sec) ka2 (1/RUs)kd2 (1/sec)Rmax (RU) Rm-obs* (RU)KD1 (nM) KD2 (nM)
RhCMVIL-101.18E+06 0.076710.0010550.0015524.320.9 65.11.3
4,617 0.1985 2.32E-040.0018724.3 0.843,000410
1.45E+04 0.21175.48E-04 0.00136 24.33.0 14,70094.4
Rate constants were obtained from fitting the Sensorgrams in Figure 6 to a bivalent analyte model.
M=molar, RU=Response unit, KD1=kd1/ka1, KD2=kd2/ka1, Rmax is the calculated Rmax determined during fitting, Rm-obs is the maximum RU value observed in
the sensorgrams at the highest concentration collected.
#Binding parameters obtained by fixing Rmax and mass transport (tc=1.546+E07) values to those obtained from global fitting of the RhCMVIL-10 data.
##Binding parameters obtained by fixing Rmax to the value obtained from global fitting of the RhCMVIL-10 data.
*The theoretical Rmax for RhCMVIL-10 dimer binding to IL-10R1 is 29.5RU.
Table 2. RhCMVIL-10 M1 and M2 Immunization Schedule.
Week ImmunogenAmountroute AmountRoute# animalsMutant
0 DNA 150 mgIM& 50 mg ID3M1
‘‘‘‘‘‘‘‘& ‘‘ ‘‘3 M2
4 DNA150 mg IM& 50 mgID3 M1
‘‘ ‘‘‘‘‘‘ & ‘‘‘‘3 M2
7 DNA150 mg IM& 50 mg ID3 M1
‘‘‘‘‘‘‘‘& ‘‘‘‘3 M2
14Protein 50 mg IM3 M1
‘‘ ‘‘‘‘‘‘3 M2
18 Protein50 mg IM3 M1
‘‘‘‘ ‘‘ ‘‘3 M2
Design and Analysis of RhCMVIL-10 Mutants
PLoS ONE | www.plosone.org7 November 2011 | Volume 6 | Issue 11 | e28127
and C viruses [40,41,42]), bacteria (e.g., M tuberculosis , C.
trachomatis , and L. monocytogenes ), protozoa (e.g., Leish-
mania [46,47], Plasmodium ), and fungi (e.g., Paracoccidioides
brasiliensis ) have all coopted activation of IL-10R1 to facilitate
the establishment and maintenance of a persistent infection, often
in conjunction with pathogenic outcomes in the infected host .
While strategies may vary between organisms, they involve the
immunosuppressive properties of either the host cIL-10, or a
pathogen-encoded IL-10 protein, to enable microenvironments of
immune privilege, tolerance, and/or immune suppression. Be-
cause of this convergent point of pathogenesis, there is now interest
in prevention and therapeutic modalities that control IL-10/IL-
10R1 engagement to maintain or restore immunity to various
Our immunization strategy takes advantage of the protracted
co-evolutionary relationship between CMV and their particular
host, in which the viral IL-10 genes underwent extreme genetic
drift from the cIL-10 gene. As a result, HCMV and RhCMV IL-
10 proteins share only 23-26% identity with their host’s cIL-10
. The extent of genetic drift in the viral orthologs is highlighted
Figure 7. Stimulation of RhCMVIL-10 binding and neutralizing antibodies (Ab) following immunization of RhCMV-infected rhesus
macaques. (A) RhCMVIL-10-binding antibodies were analyzed throughout the immunization schedule (Table 2) by ELISA measured at absorbance at
450 nm (see Methods for details). (B) Neutralization of RhCMVIL-10 WT biological activity by plasma collected throughout the immunization schedule
(Table 2). Percent (%) neutralization of RhCMVIL-10 denotes the ratio ([ IL-12 ]Plasma+RhCMVIL-10/ [ IL-12 ]Plasma)*100, such that 100% corresponds to
complete inhibition of RhCMVIL-10 and 0% is no inhibition. Values greater than 100 reflect errors/variations in the measured levels of IL-12 in the two
samples. The times of DNA (cyan) and protein (red) vaccination are shown on the Figure with arrows. Three animals (A1–A3) were immunized with
M1 (solid lines), and three animals (A4–A6) were immunized with M2 (dashed lines). The times of blood draws are noted by the solid symbols for M1
immunized animals and open symbols for M2 immunized animals. Where immunization and blood draws were performed on the same day, blood
was taken prior to immunization. The shape of the symbols denotes different animals, with M1 immunized animals A1–A3 solid diamond, square, and
circle, respectively. M2 immunized animals A4–A6 are denoted by open square, diamond, and circle, respectively. The same designations are used for
panels A and B.
Design and Analysis of RhCMVIL-10 Mutants
PLoS ONE | www.plosone.org8 November 2011 | Volume 6 | Issue 11 | e28127
by the facts that rhesus and human cIL-10 proteins share ,95%
identity, and the viral IL-10 orthologs are as divergent from each
other as they are from the cIL-10 of their host. Despite sharing low
amino acid identity, both CMVIL-10 and RhCMVIL-10
sequences are highly stable (95% identity) amongst different
strains of HCMV and RhCMV, respectively . For yet
unknown reasons, virally captured cIL-10 has drifted from what
was once a ‘self’ protein, expressed in the context of viral infection,
to one that is now highly recognizable by the host immune system.
This is highlighted by our finding that stimulation of RhCMVIL-
10-Nabs does not stimulate cross-reactive antibodies against RhIL-
10 that neutralize its biological activity (Fig 8).
Importantly, studies with LCMV, demonstrate that pathogen-
specific immune responses are not hardwired during priming but
are alterable and responsive to continuous instruction from the
antigenic environment . Like LCMV, MCMV does not
encode a cIL-10 ortholog, but stimulates production of cIL-10
. MCMV-induced up-regulation of cIL-10 impairs MHC class
II antigen presentation , antigen-specific T cell expansion
, and clearance of virally-infected cells from sites of
persistence, such as the salivary glands (SG) . Blockade of
IL-10 signaling by treatment with an antibody to IL-10R1 in
MCMV-infected mice leads to CD4 T cell-mediated clearance of
infected cells in the SG , the expansion of functional MCMV-
specific CD8 T cells and, notably, reduction of viral loads in the
spleen and lung . The observation that MCMV persistence
can be reduced by altering IL-10R1 signaling is very similar to
what has been shown for LCMV [33,57]. Thus, there are
precedents for determining whether CMVIL-10-based vaccine
therapies in an HCMV-infected host can significantly reduce long-
term parameters of HCMV infection/re-infection.
As summarized above, CMVIL-10 represents an attractive
vaccine target as it exhibits potent immunosuppressive functions
itself and induces cIL-10 in some cell types. In addition, because
of its low sequence identity with cIL-10, it is unlikely that
RhCMVIL-10-NAbs will be cross reactive with cellular IL-10.
This point has now been confirmed by experiments demonstrat-
ing that our immunized animals do not exhibit antibodies that
neutralize cellular RhIL-10 biological activity (Fig 8). However,
a remaining problem of including CMVIL-10 in any vaccination
strategy is the potential negative impact of its immunosuppres-
sive activity, which could endanger the vaccinee and/or prevent
an effective immune response to other antigens contained in the
vaccine. Crystal structure analysis has provided detailed images
of the HuIL-10/IL-10R1 and HuCMVIL-10/IL-10R1 interfac-
es required for the initial activation of the IL-10 signaling
pathway [21,27,58]. These structural studies demonstrate cIL-10
and CMVIL-10 share common and essential molecular mech-
anisms to engage the IL-10R1 chain. In addition, structural
studies have also characterized the neutralizing anti-IL-10
antibody (9D7) bound to cIL-10 . The 9D7/cIL-10 structure
illustrates that neutralization of IL-10 can occur by multiple
mechanisms in addition to steric blocking of the IL-10/IL-10R1
Based on the principles learned from the structural analyses
outline above, we have engineering non-functional RhCMVIL-10
mutants (M1, and M2) that generate antibodies that neutralize
WT RhCMVIL-10 biological activity, do not cross react with
cellular RhIL-10, and do not appear to harm the animals. These
results provide a rationale for testing this vaccination strategy in
seronegative animals, followed by challenge with RhCMV. If
experiments in the RhCMV infection model are successful, the
outcome of these experiments is directly translatable to human
studies. In particular, residues changed in RhCMVIL-10 to create
M1 and M2 are conserved in CMVIL-10. Thus, identically
mutated CMVIL-10 antigens can be made for testing in phase I
Figure 8. RhCMVIL-10-NAbs generated against RhCMVIL-10 M1 and M2 do not cross react with cellular RhIL-10. Plasma from animals
(A1–A6) pre- (pre-Imm, week 0, Figure 7), and post-immunization (week 19 plasma sample, 1 week after the second protein boost, Figure 7), was
tested for its ability to neutralize cellular RhIL-10 (grey bars) or RhCMVIL-10 (black bars) bioactivity, where bioactivity was measured as suppression of
LPS-induced IL-12 production in rhesus PBMC. The data show the biological activity of RhIL-10 is not neutralized by plasma from animals immunized
with RhCMVIL-10 M1 or M2. However, RhCMVIL-10 biological activity, except for A6, is almost completely neutralized by the same plasma sample.
Percent (%) neutralization is calculated as described in Figure 7 and in the ‘‘Materials and methods’’ section.
Design and Analysis of RhCMVIL-10 Mutants
PLoS ONE | www.plosone.org9 November 2011 | Volume 6 | Issue 11 | e28127
Materials and Methods
The University of California, Davis (UC Davis) is accredited by
the Association for Assessment and Accreditation of Laboratory
Animals Care (AAALAC, Animal Assurance #: A3433-01), a
private, nonprofit group that promotes the humane treatment of
animals in science through voluntary accreditation. UC Davis is
one of more than 640 research institutions and other organizations
that have earned AAALAC accreditation, demonstrating its
commitment to responsible animal care and use. In addition, the
CNPRC receives unannounced inspections by the U.S. Depart-
ment of Agriculture, as required by the Animal Welfare Act, and
inspections by the Food and Drug Administration as well. This
study was carried out in strict accordance with the recommenda-
tions in the Guide for the Care and Use of Laboratory Animals of
the National Institutes of Health and in accordance with the
recommendations of the Weatherall report, "The use of non-
human primates in research". All animal protocols were approved
in advance by the Institutional Animal Care and Use Committee
of UC Davis, which is fully accredited by the AAALAC. Multiple
veterinarians and animal care technicians provided state-of-the-art
care and research support for these studies. The animals were
monitored by veterinarians and trained animal care staff every day
and during all procedures. Animals were anesthetized with
ketamine during blood collection and immunization to prevent
any suffering. Care was taken to ensure that the animals were
adequately sedated under all conditions, as assessed by the
veterinarian, animal care staff, and investigators. The specific
animal use protocol for this study is 15137.
Sequence analysis and molecular modeling
Cellular and viral IL-10 amino acid sequences were aligned
using clustalW2 (EMBL-EBI). All structure analyses and figures
were generated using pymol . Cellular IL-10 and CMVIL-10
numbering is according to the alignment of the mature cIL-10
sequence, where cIL-10 residue Ser-19 in the full length sequence
is Ser-1 in this numbering scheme (Fig 2A).
Cloning, expression, and purification of RhCMVIL-10 and
The RhCMVIL-10 open reading frame (Fig 2A) of RhCMV
strain 68-1, described by Lockridge et al. , was amplified by
PCR and inserted into the pMTA-V5-His6 vector (Invitrogen) for
expression in Drosophila S2 cells (Invitrogen), as previously
described . PCR was also used to construct plasmids
containing N- (pMT-H6Fxa-RhCMVIL-10) and C-terminal
(pMT-RhCMVIL-10-FxaH6) histidine tags for affinity purification
and analysis of RhCMVIL-10 mutants. All mutations were
performed using the QuikChange site-directed mutagenesis kit
(Stratagene) and confirmed by DNA sequencing.
Transient and stable transfections of pMT-RhCMVIL-10
plasmids into Drosophila S2 cells were performed using the
manufacturers protocols (Invitrogen). Expression was induced by
addition of 0.5 mM Cu2SO4to the media and allowed to proceed
for six days. WT RhCMVIL-10 was purified by affinity
chromatography using human IL-10R1 beads, as previously
described by Jones et al. . Large scale purification of
RhCMVIL-10 mutants for vaccination studies was performed
using a 2-step nickel affinity purification protocol. Expression
media (1 Liter) was dialyzed against binding buffer (20 mM Tris
pH 8.0, 500 mM NaCl, and 5 mM imidazole) and bound to a
5 mL column of Ni-NTA resin (Novagen). RhCMVIL-10 mutant-
containing fractions were eluted from the column using bind
buffer containing 200 mM imidazole. These fractions (,30 mL)
were dialyzed against binding buffer and purified again over a
0.5 mL Ni-NTA column.
Size exclusion chromatography
Size exclusion chromatography was performed by injecting
protein samples onto a 24 mL Superdex 200 gel filtration column
(GE Health Care). Fractions (0.5 mL) were collected and analyzed
RhCMVIL-10 Pull down assay
Drosophila media (500 mL), containing transiently expressed
RhCMVIL-10 proteins, was incubated with 15 mL of IL-10R1-
coupled agarose beads (affi-gel 10, BioRad) for 1 hour at 4uC. The
beads were recovered from the media by spinning at 400xg and
washed 3 times in 500 mL of wash buffer consisting of 20 mM
Tris-HCL, pH 8.0, 150 mM NaCl, 1% Tween-20. Protein bound
to 15 mL of the washed beads was added to sample buffer, boiled 5
minutes, and loaded onto a 12% SDS-PAGE gel. Protein was
detected using coomassie staining.
RhCMVIL-10 expression media (10 mL) was run on a 12%
SDS-PAGE gel. RhCMVIL-10 proteins were detected by western-
blotting using a primary mouse anti-tetrahis antibody (Ab)
(Qiagen), diluted 1:2000 in Tris-buffered saline with Tween-20
(TBST) and 3% bovine serum albumin (BSA), followed by a sheep
anti-mouse FC horseradish peroxidase (HRP) secondary Ab,
1:5000 dilution, in TBST and 1% non-fat milk (Amersham) for
TF-1/IL-10R1 Cellular proliferation assay
TF-1/IL-10R1 cells are a human erythroleukemic cell line
transfected with the human IL-10R1 chain, which causes them to
proliferate upon addition of IL-10 to the media, which provides a
quantitative readout of IL-10 biological activity. CIL-10 and
RhCMVIL-10 mutants were dispensed into 96-well microplates
(Becton Dickinson) in duplicate wells and serially diluted three-fold
across the plates. 5,000 TF-1/IL-10R1 cells  were added to
each well and incubated for 2 days at 37uC with 5% CO2. Viable
cells were assayed using alamarBlue (Biosource International/
Invitrogen). Fluorescence intensity was measured at room
temperature using a POLARstar plate reader (BMG Lab
technologies) at wavelengths of 544 nm excitation and 590 nm
Characterization of RhCMVIL-10 mutant activity on
Ten-fold serially diluted RhCMVIL-10 M1 and M2 (0.1–
1,000 ng) were incubated in duplicate with 46105Ficoll-purified
PBMC/well in a 96 well U-bottom plate (Falcon) for 30 minutes in
a humidified 37uC incubator (5% CO2). LPS (from E. coli
O127:B8; Sigma) was then added to the cells (5 mg/mL final
concentration) followed by a 24 hour incubation at 37uC (5%
CO2). The supernatant was collected the following day and
assayed for IL-12 production by an IL-12 sandwich ELISA
IL-12 sandwich ELISA
IL-12 secretion by LPS-activated rhesus PBMC was measured
by ELISA (U-Cytech, Netherlands), according to the manufac-
turer’s protocol with slight variations. Microtitre plates (96-well,
Immulon 4 HBX, Dynex Technologies Inc.) were incubated with
Design and Analysis of RhCMVIL-10 Mutants
PLoS ONE | www.plosone.org 10November 2011 | Volume 6 | Issue 11 | e28127
IL-12 p40 and p70 capture antibodies (U-Cytech) overnight at
4uC. The plates were washed 6x with PBS containing 0.05%
tween (PBS-T) and then incubated with blocking buffer (PBS/1%
BSA) for 1 hr at 37uC. Following the block, rhesus PBMC
supernatants (100 mL/well), +/- LPS and +/-RhCMVIL-10, were
incubated overnight at 4uC. The next day the plates were washed
6 times with PBS-T, followed by the addition of 100 mL/well of
biotinylated anti-monkey IL-12 antibody for 1 hr at 37uC. After
washing, 100 mL/well of streptavidin-HRP (U-Cytech) was added
and incubated at 37uC for 1 hr. After washing, 100 mL/well TMB
substrate was added per well and the plates were incubated at
25uC for 10 min. Color development was stopped by adding
50 ml/well of 0.5 M sulfuric acid. The plates were incubated at
room temperature for 5 minutes, and then read at a wavelength of
450 nm on a Model 680 microplate reader (BioRad). Concentra-
tions of IL-12 were quantified using a 2-fold serially diluted
recombinant IL-12 standard that was included on each plate.
Characterization of RhCMVIL-10/IL-10R1 interactions by
All SPR experiments were performed on a Biacore T200 system
(GE Healthcare) at 20 uC using HBS running buffer (10 mM
Hepes (pH 7.4), 0.15 M NaCl, 0.1% P20 (GE Healthcare), and
0.1 mg/mL BSA (Sigma)). The IL-10R1-FC consisted of the
extracellular region (residues 22-235, uniprot Q13651) of the
human IL-10R1 chain fused to a mouse FC (IgG2a). The IL-
10R1-FC was captured onto a CM5 chip surface by an anti-
murine FC Ab (GE Healthcare) that was immobilized by amine
coupling to the chip as described in the manufacturer’s
instructions. IL-10R1-FC was captured on the anti-FC surface at
final densities of ,75 RU. Kinetic interaction experiments were
performed by injecting (for 90 seconds) 2-fold diluted RhCMVIL-
10 over IL-10R1-FC at a flow-rate of 75 mL/min, followed by a
150 second dissociation phase. The IL-10R1-FC surface was
regenerated by a 3 minute injection of 10 mM glycine, pH 1.7.
The data was processed and globally fit to a bivalent analyte model
(Biacore T200 Software Handbook, 2010) using Biacore T200
Software version 1.0. Estimates of RhCMVIL-10 M1 rate
constants required Rmax and tc parameters to be fixed at values
obtained while fitting the RhCMVIL-10 sensorgrams. For
RhCMVIL-10 M2, Rmax was fixed during sensorgram fitting.
Immunization of RhCMV-infected rhesus monkeys with
RhCMVIL-10 M1 and M2
Six rhesus macaques (Macaca mulatta), serologically confirmed to
RhCMVIL-10 M1 (N=3) or RhCMVIL-10M2 (N=3) using a
heterologous combination of DNA and protein. DNA vectors for
immunization were constructed by inserting the coding sequences
for RhCMVIL-10 M1 and M2 into the Sal I and Asp 718
restriction sites of the mammalian expression vector pND, which
contains the human cytomegalovirus major immediate-early
promoter and enhancer, and the SV40 polyadenylation signal
. Recombinant plasmids (pND-RhCMVIL-10 M1 and pND-
RhCMVIL-10 M2) were purified with an endotoxin-free plasmid
purification kit (QIAGEN), according to the manufacturer’s
protocol, and the DNA concentration was determined spectro-
photometrically. DNA was resuspended in PBS buffer at a
concentration of 1 mg/ml and stored at 220 uC.
Animals were immunized three times with either pND-
RhCMVIL-10 M1 or pND-RhCMVIL-10 M2 by both intrader-
mal (50 mg) and intramuscular (150 mg) injection of plasmid at
weeks 0, 4, and 7. Animals were boosted by two intramuscular
were immunizedwith either
injections of the M1 or M2 proteins at weeks 14 and 18 (50 mg)
adjuvanted in Montanide ISA 720 (Seppic Inc., Fairfield, NJ),
according to published protocols . All animal protocols were
approved in advance by the Institutional Animal Care and Use
Committee of the University of California, Davis, which is fully
accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care.
Binding antibodies against RhcmvIL-10 were characterized by
ELISA by modifying a previously published protocol . Briefly,
96-well microplates (Immulon 4 HBX, Dynex Technologies Inc.)
were coated overnight at 4uC with nickel affinity-purified
RhCMVIL-10 (12.5 ng/well) in phosphate buffered saline (PBS)
(Sigma)/0.375% sodium bicarbonate (GIBCO). Each plate was
subsequently washed 6 times with PBS/0.05% Tween 20 (Sigma)
(PBS-T) and blocked with 300 ml/well PBS/1% bovine serum
albumin (BSA) (Sigma) for 2 hours at 25uC in a temperature-
controlled incubator. After washing the plates 6 times with PBS-T,
100 ml of a 1:100 dilution of week 19 plasma (in PBS-T/1% BSA)
was added to each well and incubated at 25uC for 2 hours. The
plates were subsequently washed 6 times with PBS-T wash buffer
and loaded with 100 ml/well of a 1:60,000 dilution of peroxidase-
conjugated goat-anti-monkey IgG (Kirkegaard & Perry Laborato-
ries, Inc - KPL) and incubated at 25uC for 1 hour. The plates were
then washed 6 times with PBS-T wash buffer and 100 ml/well of
tetramethylbenzidine liquid substrate (TMB) (Sigma) was added
and incubated for 30 min at 25uC. TMB color development was
stopped by the addition of 50 ml/well of 0.5 M sulfuric acid. After
a five minute incubation at room temperature, color development
was quantified spectrophotometrically at a wavelength of 450 nm
on a Model 680 microplate reader (BioRad).
Analysis of rhesus plasma for neutralizing anti-RhCMVIL-
Rhesus macaque plasma samples, collected during immuniza-
tion (Fig. 7), were diluted (1:4,000) in RPMI/10% fetal bovine
serum and incubated in the presence, or absence, of recombinant
WT RhCMVIL-10 (0.5 ng/mL) for 3 hours at 37uC. 200 mL of
the plasma +/- RhCMVIL-10 were incubated in duplicate with
46105Ficoll-purified PBMC/well in a 96 well U-bottom plate
(Falcon) for 30 minutes in a humidified 37uC incubator (5% CO2).
LPS (from E. coli O127:B8; Sigma) was then added to the cells
(5 mg/mL final concentration) followed by a 24 hour incubation at
37uC (5% CO2). Supernatants were collected the following day
and the amount of IL-12 is each sample was assayed by ELISA as
described above. The presence of RhCMVIL-10-NAbs was
determined by comparing IL-12 levels (pg/mL) produced by
LPS-activated PBMC incubated with plasma and recombinant
WT RhCMVIL-10 (which at 0.5 ng/mL inhibits all IL-12
production in the absence of plasma) versus plasma only. The
ratio ([ IL-12 ]Plasma+RhCMVIL-10/ [ IL-12 ]Plasma)*100, is plotted
for each plasma sample, and represents the percent of recombi-
nant WT RhCMVIL-10 biological activity neutralized by each
Analysis for cross-neutralizing anti-RhIL-10 antibodies in
Rhesus macaque week 19 plasma samples were analyzed for
any cross-neutralization of RhIL-10 biological activity. The assay
was performed as described for neutralization of WT RhCMVIL-
10 biological activity, except that cells were incubated in 12.5 ng/
ml of RhIL-10, which was the minimum concentration of RhIL-10
Design and Analysis of RhCMVIL-10 Mutants
PLoS ONE | www.plosone.org 11November 2011 | Volume 6 | Issue 11 | e28127
required to suppress IL-12 production in LPS-activated Rhesus
RhIL-10 was kindly provided by Dr. Francois Villinger (Emory University)
through the Resource for Nonhuman Primate Immune Reagent Program
DNA sequencing was provided by the UAB Heflin Center Genomics
Core Facility. Usage of the Biacore T-200 is made possible by the UAB
Multidisciplinary Molecular Interaction Core (MMIC). We thank Bethany
Harris for technical assistance in protein purification.
Conceived and designed the experiments: MRW PAB. Performed the
experiments: NJL MKE CEA PAB MRW. Analyzed the data: NJL MKE
CEA PAB MRW. Wrote the paper: MRW PAB.
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