Evidence of early B-cell dysregulation in simian immunodeficiency virus infection: rapid depletion of naïve and memory B-cell subsets with delayed reconstitution of the naïve B-cell population.
ABSTRACT Despite eliciting a robust antibody response in humans, several studies in human immunodeficiency virus (HIV)-infected patients have demonstrated the presence of B-cell deficiencies during the chronic stage of infection. While several explanations for the HIV-induced B-cell deficit have been proposed, a clear mechanistic understanding of this loss of B-cell functionality is not known. This study utilizes simian immunodeficiency virus (SIV) infection of rhesus macaques to assess B-cell population dynamics beginning at the acute phase and continuing through the chronic phase of infection. Flow cytometric assessment demonstrated a significant early depletion of both naïve and memory B-cell subsets in the peripheral blood, with differential kinetics for recovery of these populations. Furthermore, the altered numbers of naïve and memory B-cell subsets in these animals corresponded with increased B-cell activation and altered proliferation profiles during the acute phase of infection. Finally, all animals produced high titers of antibody, demonstrating that the measurement of virus-specific antibody responses was not an accurate reflection of alterations in the B-cell compartment. These data indicate that dynamic B-cell population changes in SIV-infected macaques arise very early after infection at the precise time when an effective adaptive immune response is needed.
- SourceAvailable from: David C Montefiori[Show abstract] [Hide abstract]
ABSTRACT: The design of an effective vaccine to reduce the incidence of mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) via breastfeeding will require identification of protective immune responses that block postnatal virus acquisition. Natural hosts of simian immunodeficiency virus (SIV) sustain nonpathogenic infection and rarely transmit the virus to their infants, despite high milk virus RNA loads. This is in contrast to HIV-infected women and SIV-infected rhesus macaques (RhMs), non-natural hosts which exhibit higher rates of postnatal virus transmission. In this study, we compared the systemic and mucosal B cell responses of lactating, SIV-infected African green monkeys (AGMs), a natural host species, to that of SIV-infected RhMs and HIV-infected women. AGMs did not demonstrate hypergammaglobulinemia or accumulate circulating memory B cells during chronic SIV infection. Moreover, the milk of SIV-infected AGMs contained higher proportions of naive B cells compared to RhMs. Interestingly, AGMs exhibited robust milk and plasma Env binding antibody responses that were one to two logs higher than in RhMs and humans and demonstrated autologous neutralizing responses in milk at one year post infection. Furthermore, the plasma and milk Env gp120-binding antibody responses were equivalent to or predominant over Env gp140-binding antibody responses in AGMs, in contrast to that in RhMs and humans. The strong gp120-specific, functional antibody responses in the milk of SIV-infected AGMs may contribute to the rarity of postnatal transmission observed in natural SIV hosts.Journal of Virology 08/2013; · 5.08 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: SHIV models for HIV infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIVSF162P3 infection in one month-old rhesus macaques results not only in high persistent plasma viremia but also leads to very rapid disease progression within 12-16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses SHIVIG reduces both plasma and PBMC-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed in order to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The one month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance and the induction of adaptive immunity in disease progression.Journal of Virology 07/2013; · 5.08 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In pathogenic HIV and SIV infections of humans and rhesus macaques (RMs), preferential depletion of CD4(+) Th17 cells correlates with mucosal immune dysfunction and disease progression. Interleukin (IL)-21 promotes differentiation of Th17 cells, long-term maintenance of functional CD8(+) T cells, and differentiation of memory B cells and antibody-secreting plasma cells. We hypothesized that administration of IL-21 will improve mucosal function in the context of pathogenic HIV/SIV infections. To test this hypothesis, we infected 12 RMs with SIVmac239 and at day 14 post-infection treated six of them with rhesus rIL-21-IgFc. IL-21-treatment was safe and did not increase plasma viral load or systemic immune activation. Compared to untreated animals, IL-21-treated RMs showed (i) higher expression of perforin and granzyme B in total and SIV-specific CD8(+) T cells and (ii) higher levels of intestinal Th17 cells. Remarkably, increased levels of Th17 cells were associated with reduced levels of intestinal T cell proliferation, microbial translocation and systemic activation/inflammation in the chronic infection. In conclusion, IL-21-treatment in SIV-infected RMs improved mucosal immune function through enhanced preservation of Th17 cells. Further preclinical studies of IL-21 may be warranted to test its potential use during chronic infection in conjunction with antiretroviral therapy.PLoS Pathogens 07/2013; 9(7):e1003471. · 8.14 Impact Factor
JOURNAL OF VIROLOGY, Mar. 2010, p. 2466–2476
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 5
Evidence of Early B-Cell Dysregulation in Simian Immunodeficiency
Virus Infection: Rapid Depletion of Naïve and Memory
B-Cell Subsets with Delayed Reconstitution of the
Naïve B-Cell Population?
David Kuhrt,1,2Seth A. Faith,1,2Amanda Leone,3Mukta Rohankedkar,4
Donald L. Sodora,3Louis J. Picker,4and Kelly Stefano Cole1,2*
Department of Immunology1and Center for Vaccine Research,2University of Pittsburgh, Pittsburgh, Pennsylvania 15261;
Seattle Biomedical Research Institute, Seattle, Washington 981093; and Vaccine and Gene Therapy Institute,
Oregon Health and Science University, Beaverton, Oregon 970064
Received 16 September 2009/Accepted 9 December 2009
Despite eliciting a robust antibody response in humans, several studies in human immunodeficiency virus
(HIV)-infected patients have demonstrated the presence of B-cell deficiencies during the chronic stage of
infection. While several explanations for the HIV-induced B-cell deficit have been proposed, a clear mecha-
nistic understanding of this loss of B-cell functionality is not known. This study utilizes simian immunodefi-
ciency virus (SIV) infection of rhesus macaques to assess B-cell population dynamics beginning at the acute
phase and continuing through the chronic phase of infection. Flow cytometric assessment demonstrated a
significant early depletion of both naïve and memory B-cell subsets in the peripheral blood, with differential
kinetics for recovery of these populations. Furthermore, the altered numbers of naïve and memory B-cell
subsets in these animals corresponded with increased B-cell activation and altered proliferation profiles during
the acute phase of infection. Finally, all animals produced high titers of antibody, demonstrating that the
measurement of virus-specific antibody responses was not an accurate reflection of alterations in the B-cell
compartment. These data indicate that dynamic B-cell population changes in SIV-infected macaques arise very
early after infection at the precise time when an effective adaptive immune response is needed.
Effective B-cell responses result in the generation of mem-
ory B-cell populations which are able to proliferate and pro-
duce antibodies that can control primary and secondary insults
by microbial pathogens (2). Impaired maturation and timing of
B-cell-mediated immune responses result in the production
of ineffective antibodies, which are unable to control infec-
tion and may result in the persistence of the pathogen (36).
Although human immunodeficiency virus (HIV) infection
generally elicits high-titer antibodies, virus-specific titers do
not correlate with delayed clinical progression, suggesting
that antibodies produced during HIV infection are not suffi-
cient to provide long-term viral control (6). Ineffective anti-
body production in the context of HIV infection could be a
result of numerous T-cell and B-cell abnormalities induced
either directly or indirectly through infection. B-cell perturba-
tions, characterized during chronic infection, include hyper-
gammaglobulinemia (11, 31), a diminished in vitro response to
mitogenic stimulation (10, 37), diminished antibody responses
to vaccination (15, 23), and loss of memory B-cell subsets (3,
10, 37). It is highly likely that these B-cell abnormalities are
linked with the inability of HIV-infected individuals to form
effective antibody responses to HIV and opportunistic patho-
B-cell perturbations during acute HIV infection may lead
to dysfunctions observed during chronic infection. Despite
numerous reports that hypothesized that B-cell phenotypic
and functional abnormalities arise due to the effects of
chronic infection, a limited number of acute infection stud-
ies have provided evidence that B-cell dysfunctions may be
initiated much earlier. Studies by De Milito et al. and others
have reported a decrease in CD27?B cells associated with
chronic HIV infection (3, 4, 10–12, 15, 30, 31, 36–38, 40). The
reduction of this population may explain the diminished anti-
body responses to non-HIV antigens present in HIV-infected
individuals. However, the mechanism for this loss of memory B
cells during chronic infection is unclear. One possibility is that
B-cell losses are related to reduced T-cell numbers. In a study
by Titanji et al., a strong correlation between the number of
CD4 T cells and the percentage of memory B cells was re-
ported in chronic HIV infection (37). Conversely, others have
reported that no correlation was found between CD4 numbers
and memory B-cell numbers (3, 10). Interestingly, reductions
in percentages of B cells, increased expression of Fas on B
cells, increased total plasma IgG levels, a decreased percentage
of IgM memory B cells, and decreased B-cell responses to
antigenic stimulation have been shown to occur within 6
months of HIV infection (36, 37). Disruption of germinal cen-
ters in the gut during acute HIV infection may also compro-
mise the humoral immune response (20). While these studies
provide insight into virus-induced changes in the B-cell com-
partment during infection, it is difficult to ascertain precisely
when these changes occur, due to limitations in sample size
and numbers during this early period of infection. The con-
* Corresponding author. Mailing address: 3501 Fifth Avenue, 8035
Biomedical Science Tower 3, Pittsburgh, PA 15261. Phone: (412) 648-
8685. Fax: (412) 648-8721. E-mail: firstname.lastname@example.org.
?Published ahead of print on 23 December 2009.
flicting reports reflect the high amount of variability present in
human HIV infection and illuminate the need for a model to
study B-cell populations in which experimental parameters can
be more rigorously controlled. An understanding of the effects
of HIV on the B-cell population during this critical early phase
of infection is needed to determine how the initial interactions
between virus and host immune system set the stage for long-
term disease progression in the infected host. The simian im-
munodeficiency virus (SIV)/macaque model provides a system
in which to ask these questions.
Studies in SIV-infected macaques have demonstrated that
the number of total B (CD20?) cells in the periphery decreases
dramatically during the acute phase of infection (13, 24). The
loss of these cells coincides with a similar depletion of periph-
eral CD4 T cells and is associated with primary viremia. Inter-
estingly, the loss of total B cells is greater in magnitude than
the loss of CD4?T cells (24). In order to understand how these
cells are being depleted, it is necessary to characterize B-cell
subsets during SIV infection in the macaque. The present study
was designed to assess phenotypic changes in B-cell numbers
during the acute phase of SIV infection, both in the total B-cell
population as well as in B-cell subsets. Our results identified
early, rapid changes in B-cell subsets that were not apparent in
analysis of the total B-cell population. Specifically, we identi-
fied a significant depletion from the periphery of both the
naïve (CD20?CD27?) and memory (CD20?CD27?) B-cell
populations during acute infection and increased total B-cell
population activation that may be related to ineffective anti-
body production commonly associated with SIV infection. Fur-
thermore, the data demonstrate that measurement of enve-
lope-specific antibody responses was not a sensitive reflection
of SIV effects on B-cell subsets. These data provide novel
information about the timing and dynamics of phenotypic
changes in the B-cell compartment during SIV infection that
may be associated with functional changes observed later in
chronic infection. These results can be used to tailor therapeu-
tic treatments designed to preserve the B-cell compartment
early in SIV/HIV infection.
MATERIALS AND METHODS
Animals. Ten colony-bred rhesus macaques (Macaca mulatta) of Indian origin
were maintained and used in accordance with the guidelines of the Animal Care
and Use Committee at the Oregon National Primate Research Center. Animals
were infected on day zero with SIVmac239 intravenously with 10,000 infectious
units. Beginning at day 105 postinfection all animals received daily antiretroviral
therapy (ART) which included both tenofovir (PMPA; 30 mg/kg of body weight
until day 134 and then 20 mg/kg subsequently) and emtricitabine (FTC; 50 mg/kg
until day 134 and then 20 mg/kg subsequently). Complete blood counts were
obtained at each blood draw by using a Coulter ACT 5 Diff open reader cell
counter (Beckman Coulter, Fullerton, CA).
Viral quantification. Assessment of plasma SIV RNA was carried out using a
real-time reverse transcription-PCR assay (threshold sensitivity ?100 SIV gag
RNA copy equivalents/ml of plasma; interassay coefficient of variation ?25%)
Flow cytometric analysis. Flow cytometric analysis was performed on fresh
lysed whole blood samples as described previously (39). Briefly, 100 ?l of citrate
treated whole blood was obtained in a blood collection tube (BD Diagnostics,
Franklin Lakes, NJ). Biotinylated or directly fluorochrome-conjugated antibod-
ies to extracellular targets were incubated with whole blood at room temperature
for 60 min. Following incubation, cells were washed once with 4 ml of cold (4°C)
phosphate-buffered saline (PBS) with 0.1% of bovine serum albumin and 0.02%
sodium azide (wash buffer). Cells were then fixed and permeabilized by a 10-min
incubation at room temperature with fluorescence-activated cell sorting (FACS)
lysing solution (BD Biosciences, San Jose, CA), washed, and incubated twice
with 0.5 ml of FACS permeabilizing solution (BD Biosciences, San Jose, CA) for
10 min at room temperature. Cells were washed twice with cold wash buffer and
then stained with directly conjugated intracellular antibodies at room tempera-
ture for 30 min, washed once, and analyzed by flow cytometry. Freshly stained
lymphocytes were differentiated using forward and side scatter characteristics on
an LSRII apparatus (BD Biosciences, San Jose, CA).
The total B-cell population was differentiated using CD20 (eBioscience, San
Diego, CA) expression and was further subdivided according to expression of
CD27 (BD Bioscience, San Jose, CA) and IgD (Southern Biotech, Birmingham,
AL). B cells were characterized as CD20?CD27?(memory) or CD20?CD27?
(naïve). Further parsing of memory cells using IgD identified IgM-secreting
(CD20?CD27?IgD?) cells as well as IgG/IgA-secreting (CD20?CD27?IgD?)
cells. Cell populations were also assessed for surface expression of CD95 (BD
Bioscience) and Ki67 expression levels (BD Bioscience, San Jose, CA) as mea-
sures of activation and proliferation. CD4 T-cell counts were obtained by ana-
lyzing expression of CD3-positive (BD Bioscience, San Jose, CA) and CD4-
positive (BD Bioscience, San Jose, CA) cells. List-mode multiparameter data
files were analyzed using the FlowJo software program (PC version 7.2.5; Tree
Star Inc., Ashland, OR).
SIV envelope-specific antibody endpoint titer. Antibody reactivities to deter-
gent-disrupted SIVsmB7 envelope proteins (16) were determined in a con-
conavalin A (ConA) enzyme-linked immunosorbent assay (ELISA) as previously
described (7). Briefly, SIVsmB7 viral envelope proteins (gp120 and gp41) were
captured onto 96-well microtiter plates (Immulon 2HB; Dynex Technologies,
Chantilly, VA) coated with 5 ?g of ConA/well for 1 h at 25°C. After a washing
step with PBS, nonspecific binding was blocked by the addition of 5% dry milk
in PBS (blocking solution) to all wells and incubation for 1 h at 25°C. Heat-
inactivated plasma samples were serially diluted in blocking solution and incu-
bated in the SIVsmB7 envelope-coated wells for 1 h at 25°C. After an extensive
washing, peroxidase-conjugated anti-monkey IgG (Nordic Immunology Labora-
tories, Tilburg, The Netherlands) was diluted in blocking solution, added to each
well, and incubated for 1 h at 25°C and washed. Following the final wash step, all
wells were incubated with TM Blue substrate (Seracare, Milford, MA) for 20 min
at room temperature, color was developed by the addition of 1 N sulfuric acid,
and colorimetric analysis of antibody binding to SIVsmB7 was performed at an
optical density of 450 nm (OD450) using a Spectra Max 340 PC (Molecular
Devices, Sunnyvale, CA).
Statistical analyses. Statistical analyses were performed using GraphPad
Prism 4 (San Diego, CA). Paired t tests were performed, comparing values for
individual and longitudinal time points to the time zero value within a popula-
Acute-phase decline in total B-cell levels corresponds with a
peak in viral titer and drop in CD4?T cells. Ten rhesus
macaques were infected intravenously with SIVmac239 at the
Oregon National Primate Research Center. Peripheral blood
samples were analyzed longitudinally during the first 150 days
postinfection. Viral titers peaked at day 10 postinfection
(mean, 3.5 ? 107) and declined to an average set point at day
84 (mean, 7.3 ? 106) (Fig. 1). Two animals did not control viral
replication, maintaining viral loads of 107to 108copies/ml for
100 days postinfection. Five animals demonstrated intermedi-
ate levels of control, with setpoint viral titers of 106to 107
copies/ml. The last three animals had relatively effective viral
control, with viral titers that continued to gradually decline for
about 60 to 85 days postinfection, reaching setpoint titers of
CD4?T cells decreased during the first 10 days postinfec-
tion as previously reported (24), reaching the lowest point of
approximately 800 cells/?l, coincident with the peak in viral
load (Fig. 2). Consistent with a prior study by Roederer et al.,
the peripheral CD20?B-cell population also exhibited a sub-
stantial drop in cell numbers, from an average of 1,157 cells/?l
preinfection to a nadir average of 378 cells/?l at day 10 postin-
fection (24). Following this initial drop, the number of periph-
VOL. 84, 2010 EARLY B-CELL DYSREGULATION IN SIV INFECTION2467
eral CD20?(total B) cells rebounded by 30 days postinfection
to an intermediate plateau, then recovered to preinfection
levels between 80 and 90 days postinfection. Interestingly, a
spike in total B-cell numbers was observed following the initi-
ation of ART.
Naïve (CD20?CD27?) B-cell numbers remained signifi-
cantly lower than baseline levels longer than memory (CD20?
CD27?) B-cell numbers. To further delineate naïve and mem-
ory B-cell subsets, total B cells were subdivided based on sur-
face expression of CD27 (Fig. 3). For these studies, we took
advantage of human antibodies that cross-reacted with the
rhesus system, utilizing markers previously defined in humans.
Thus, putative naïve cells were defined as cells that expressed
CD20?CD27?, while putative memory cells were defined as
CD20?CD27?and will be referred to going forward as naïve
and memory cells, respectively.
Memory (CD20?CD27?) cell numbers (Fig. 4b) declined
after day 3 postinfection to an average of 255.5 cells/?l by day
10 postinfection (Fig. 4c). The number of memory cells recov-
ered to a level that was slightly reduced from preinfection
levels (748.8 cells/?l) by day 30 postinfection. The number of
naïve (CD20?CD27?) cells (Fig. 4a) increased significantly
FIG. 1. Measurement of viral loads following SIV infection. Ten rhesus macaques were infected intravenously with SIVmac239 and analyzed
for the number of viral copies per milliliter of blood from postinfection day (PID) 0 to 149. Viral loads for each animal are plotted individually.
All animals were treated with PMPA (30 mg/kg from day 105 until day 134 and then 20 mg/kg subsequently) and FTC (50 mg/kg from day 105
until day 134 and then 20 mg/kg subsequently) (shaded area). Data for animals with high viral loads are delineated by dotted lines, intermediate
viral loads are shown by solid lines, and low viral loads are shown by dashed lines.
FIG. 2. Measurement of CD4?T-cell and total CD20?B-cell numbers following SIV infection. The average numbers of CD4?T cells and total
CD20?B cells for all animals are shown. Data represent the averages of 10 animals, with error bars representing the standard errors between the
animals for each time point. Asterisks denote a significant difference from a particular time point compared to the day zero value, using Student’s
t test (P ? 0.05).
2468 KUHRT ET AL. J. VIROL.
(P ? 0.005) from an average of 401.4 cells/?l at day 0 to 519.2
cells/?l at day 3 (Fig. 4c and Table 1). This initial increase was
then followed by a significant decrease in cell numbers, reach-
ing the lowest point by day 10 postinfection. The naive cell
population remained significantly diminished compared to pre-
infection values out to day 40 postinfection, only returning to
preinfection levels between days 80 and 90 postinfection.
Incomplete recovery of IgM-secreting (CD20?CD27?IgD?)
cells. Memory B cells were further differentiated based upon
surface IgD expression (26, 27). In humans, CD20?CD27?
IgD?cells have been found to predominantly secrete IgM
antibodies (34), while CD20?CD27?IgD?cells have been
found to predominantly secrete IgG or IgA antibodies (1).
Using these markers in the rhesus system, both IgM-secreting
(Fig. 5a) and IgG/IgA-secreting (Fig. 5b) cell populations de-
creased in total numbers of cells within 3 to 10 days postinfec-
tion. Following the initial decrease in cell number, IgG/IgA-
secreting cells rapidly increased by day 28, to a mean of 303.8
cells/?l, where they remained steady until day 70 (Fig. 5c and
Table 2). After day 70 this population once again increased,
returning to preinfection values by day 96 postinfection. In
contrast, IgM-secreting cells demonstrated a gradual increase
in cell numbers between days 10 and 28 postinfection followed
by a gradual decline after day 40 (Fig. 5c). Interestingly, IgM-
secreting cells failed to rebound to preinfection values until the
initiation of ART.
Dramatic and early increases in CD95 expression in all
B-cell subsets. To assess whether differences in B-cell recovery
between memory subsets were due to increased cell activation,
surface expression of CD95 was analyzed. All B-cell popula-
tions demonstrated increased CD95 surface expression follow-
ing infection, despite differences in the basal level of CD95
expression. The magnitudes of the increases were also different
between B-cell subsets (Fig. 6). For example, the lowest level
of basal surface expression of CD95 (1.64%) was observed in
the naïve B-cell population, and a significant increase in the
percentage of these cells expressing CD95 (3.62%) (P ? 0.05)
was observed by day 7 postinfection (Fig. 6, closed circles). The
percentage of CD95?naïve B cells remained significantly ele-
vated above baseline from day 7 through the entire study
period. IgM-secreting cells expressed moderate basal levels of
CD95 (17.9%) with a significant increase in the percentage of
CD95-expressing cells to 27.0% by day 7 postinfection (P ?
0.05). The percentage of IgM-secreting cells expressing CD95
remained significantly higher than preinfection levels for the
duration of the study period, with the maximum percentage
observed being 44.8% (Fig. 6, open triangles). Finally, 66.1%
of IgG/IgA-secreting cells expressed CD95 preinfection (Fig. 6,
closed squares), with a significant increase in expression (P ?
0.05) by day 3 postinfection. As seen in the other populations
studied, the increased expression remained significantly ele-
vated for the duration of the study, with a maximum of 86.5%
of IgG/IgA-secreting cells expressing surface CD95.
Contrasting proliferative responses to SIV infection be-
tween B-cell subsets. To assess whether decreased prolifera-
tion played a role in acute B-cell depletion, B-cell subsets were
further analyzed for Ki67 expression. The proliferative re-
sponses observed were remarkably different when comparing
the naïve, IgM-secreting and IgG/IgA-secreting populations
(Fig. 7). The percentage of Ki67?naïve cells slowly decreased
for the entire duration of the study from 15.1% preinfection to
4.8% at day 149. ART did not alter the decline in the percent-
age of Ki67?cells in this naïve B-cell population. In contrast,
memory B cells exhibited a spike in the percentage of Ki67?
cells at day 21. The IgG/IgA-secreting population demon-
strated a significant increase from 16.6% preinfection to 40.2%
at day 21 (P ? 0.005), followed by a moderate decline, with the
average steady-state level of Ki67?cells being maintained at a
higher level than preinfection until the initiation of ART. A
significant increase in the percentage of Ki67?IgM-secreting
cells was observed between preinfection and day 21 (19.5% to
32.2%, respectively; P ? 0.05). This increase was followed by a
significant decline, with the percentage of cells being main-
tained at a lower level than preinfection levels until initiation
Differential effects of ART treatment on B-cell subsets.
Treatment with PMPA (50 mg/kg) and FTC (30 mg/kg) was
initiated at day 105 postinfection in all animals. The dosage of
FIG. 3. Differentiation of B-cell subsets using flow cytometric analysis. Representative plots of whole blood FACS staining and B-cell
differentiation using antibodies to CD20, CD27, and IgD are shown. (a) CD20?B cells were differentiated from the total lymphocyte population
and delineated using side and forward scatter characteristics. (b) Expression of CD27 on B cells was used to separate CD27?(naïve) from CD27?
(memory) B cells. (c) Memory B cells were further subdivided based on expression of IgD into CD20?CD27?IgD?(IgM-secreting) and CD20?
VOL. 84, 2010EARLY B-CELL DYSREGULATION IN SIV INFECTION 2469
FIG. 4. Longitudinal analysis of naïve (CD20?CD27?) and memory (CD20?CD27?) B cells. Peripheral blood from rhesus macaques infected
with SIVmac239 was obtained at the indicated time points after acute and early chronic infection. Cells were stained for surface expression of CD20
and CD27 and analyzed by flow cytometry to differentiate naïve and memory populations. Data representing the number of CD20?CD27?naïve
B cells (a) or CD20?CD27?memory B cells (b) over longitudinal time points are shown. The mean value for all 10 animals in each group is
indicated by the dark solid line. (c) Average numbers of naïve (CD20?CD27?) and memory (CD20?CD27?) B cells, with error bars representing
standard errors for all 10 animals. Data during ART are indicated by the shaded area. Asterisks denote a significant difference from a particular
time point compared to the day 0 value, using Student’s t test (P ? 0.05).
PMPA was reduced at day 134 to 20 mg/kg, and at the same
time the dosage for FTC was reduced to 20 mg/kg. Following
initiation of ART, all animals demonstrated an initial drop in
viral setpoint, with the noncontrollers and intermediate con-
trollers rebounding within an average of 15 to 25 days after
treatment, respectively. In contrast, the three animals demon-
strating the lowest viral setpoint had the most dramatic drop
following initiation of ART, with two of the three animals
reaching undetectable levels of virus within 10 days after treat-
ment and the third animal’s levels becoming undetectable
within 45 days of treatment initiation (Fig. 1). Antiretroviral
therapy resulted in a slight increase in the number of total B
cells (Fig. 2). However, changes within B-cell subsets were
more dramatic. The naïve B-cell population demonstrated an
initial, moderate increase in cell numbers following the initia-
tion of ART (Fig. 4), from 408 cells/?l at day 105 to 562.6
cells/?l at day 112. The increase in naïve B-cell numbers at the
initiation of ART was then followed by a steady decline to 298
cells/?l by day 149. Memory B cells also demonstrated pro-
nounced increases in cell numbers (Fig. 4), from 571 cells/?l at
day 105 to 954.3 cells/?l at day 122. In contrast to the naïve
B-cell population, the number of memory cells remained
higher than pretreatment numbers out to day 149. Interest-
ingly, ART did not elicit changes in the surface expression of
CD95 on any of the B-cell populations studied (Fig. 6). While
ART had no effect on the Ki67 expression in the naïve B-cell
population, it did result in a transient increase in the percent-
age of IgM-secreting B cells expressing Ki67 and a sustained
decrease in the percentage of IgG/IgA-secreting cells express-
ing Ki67 (Fig. 7).
Antibody production during early SIV infection. To deter-
mine how analysis of B-cell subsets correlated with the pro-
duction of SIV-specific antibody production, SIV envelope-
specific antibody endpoint titers were measured in longitudinal
serum samples in a concanavalin A ELISA. All animals with
the exception of one rapid progressor demonstrated high-titer
SIV-specific antibody by 4 weeks postinfection, and these titers
were sustained for the duration of the study (Fig. 8). Samples
from early time points (days 0 to 56) were not available for
endpoint titer analysis. However, results were consistent with
data previously published from our laboratory demonstrating
that antibody titers from historical controls infected with SIV
rapidly rise and peak within 4 to 12 weeks postinfection (6, 7).
This study is, to our knowledge, the first detailed, longitudi-
nal analysis of B-cell subsets during acute and early chronic
SIV infection. Novel changes were identified very rapidly fol-
lowing SIV infection within specific B-cell subsets that have not
been observed in previous studies when analyzing total B cells.
While all B-cell subsets in this study demonstrated a similar
and profound depletion in cell number concordant with peak
viremia, differences in the timing of recovery to preinfection
values were observed among the different populations. The
depletion of multiple B-cell subsets from the periphery during
acute infection may indicate a compromise in the early B-cell
response to SIV. Further, these depletions may play a role in
abnormal B-cell maintenance and functionality observed dur-
ing later stages of infection For example, from these studies it
is not clear whether the B cells are depleted due to direct or
indirect viral effects or whether cells are trafficking from the
periphery to tissues. Additional studies to determine the mech-
anisms for this depletion are needed.
It is likely that a combination of B-cell depletion and redis-
tribution to lymphoid organs occurs during acute SIV infec-
tion. The current study only addressed B-cell populations in
the periphery and could not directly demonstrate whether de-
pletions from the periphery were reflected in the spleen or
lymph nodes. Prior studies in cynomolgus and rhesus ma-
caques indicated that redistribution of a portion of B cells to
the spleen and lymph nodes occurs during acute infection (32,
42). Germinal centers have been shown to be reservoirs for
virus and are areas where virus and viral proteins are likely to
interact with B cells (42). As such, interactions in lymphoid
organs may also lead to B-cell dysfunction, as there is evidence
that the presence of proliferating B cells within the germinal
centers of lymph nodes decreases as early as day 20 postinfec-
tion (42). Thus, the depletion of peripheral B cells from the
periphery, either via redistribution to lymphoid organs or cell
death, would have a detrimental effect on the B-cell popula-
Several B-cell functional abnormalities have been associated
with HIV infection, including hypergammaglobulinemia (3, 8,
10, 11, 31, 37, 41), increased basal activation accompanied by
diminished reactivity to mitogenic stimulation (11, 14, 26–28,
31), and depletion of the memory B-cell subset (3, 10, 11, 15,
TABLE 1. Changes in CD20?CD27?(naı ¨ve) and CD20?CD27?
(memory) B-cell subsets compared to baseline
following SIV infection
No. of cells
No. of cells
0 750.59 (527.57)
aNumber of days postinfection with SIVmac239 (10,000 infectious units, in-
bNumber of CD20?CD27?(naı ¨ve) B cells present in peripheral blood at the
indicated time point; values represent means of 10 animals.
cPercent change in CD20?CD27?(naı ¨ve) B-cell number at each time point
compared to the day zero preinfection time point.
dNumber of CD20?CD27?(memory) B cells present in peripheral blood at
indicated time points; values represent the mean of 10 animals.
ePercent change in CD20?CD27?(memory) B cell number at each time
point compared to the day zero preinfection time point.
fInitiation of ART (PMPA and FTC).
VOL. 84, 2010 EARLY B-CELL DYSREGULATION IN SIV INFECTION2471
FIG. 5. Longitudinal analysis of IgG/IgA-secreting (CD20?CD27?IgD?) and IgM-secreting (CD20?CD27?IgD?) cells. Peripheral blood from
rhesus macaques infected with SIVmac239 was obtained at the indicated time points after acute and early chronic infection. Cells were stained with the
surface markers CD20, CD27, and IgD and analyzed by flow cytometry to differentiate IgG/IgA-secreting and IgM-secreting cells. (a and b) Data
representing the number of CD20?CD27?IgD?IgG/IgA-secreting B cells (a) or CD20?CD27?IgD?IgM-secreting B cells (b) over longitudinal time
points. The mean value for all 10 animals in each group is indicated by the dark solid line. (c) Average numbers of IgG/IgA-secreting (CD20?CD27?
IgD?) and IgM-secreting (CD20?CD27?IgD?) B cells, with error bars representing standard errors for all 10 animals. Data during ART are indicated
by the shaded area. Asterisks denote a significant difference from the day zero value, based on Student’s t test (P ? 0.05).
30, 31, 37). However, due to the difficulty in obtaining longi-
tudinal samples from HIV-infected patients, especially during
the early stages of infection, studies in HIV-infected patients
have predominantly represented a snapshot of the B-cell rep-
ertoire at one or very few time points during the chronic phase
of infection. Host-virus interactions that occur during the acute
phase of HIV infection are known to heavily influence disease
progression (36). Thus, the SIV/macaque model provides an
effective means for a longitudinal B-cell analysis during acute
The present study identified dynamic changes in the memory
B-cell population during acute infection. The number of mem-
ory B cells within the periphery significantly dropped within 7
days of infection. The IgG/IgA-secreting B-cell population re-
covered to a level just below preinfection numbers within 30
days, while the IgM-secreting B-cell population recovered
more slowly. The human B-cell population which is analogous
to the IgM-secreting B-cell population in rhesus macaques has
been shown to be critical for the production of antibodies to
newly emerging viral mutants and opportunistic infections and
is also very important in the production of T-cell-independent
responses to pathogens, including pneumococcus (17–19, 22).
Thus, a slow recovery in this B-cell subset has the potential to
slow the initial response to SIV/HIV during acute infection
and to render the host more susceptible to opportunistic in-
fections later on. Defining the mechanism underlying the loss
of this B-cell subset could aid in the design of targeted immune
therapy to protect the host from opportunistic infections.
Measurement of total antibody production has been a gold
standard by which investigators measure the functionality of
the B-cell compartment. However, data from our lab and oth-
ers have demonstrated that the production of quantitative lev-
els of virus-specific antibodies may not necessarily indicate that
the antibody produced is qualitatively effective at limiting virus
replication (6, 7, 29, 35). Thus, the functional relevance of the
antibody response in controlling infection may still be compro-
mised despite measurement of a robust antibody titer. In a
recent study by Scheid et al., IgG clones derived from memory
B cells in HIV-infected patients demonstrating broad neutral-
FIG. 6. Expression of CD95 in B-cell subsets following SIVmac239 infection. The average percentages of IgG/IgA-secreting (closed squares),
IgM-secreting (open triangles), and naïve (closed circles) B cells expressing CD95 were followed in longitudinal peripheral blood samples from 10
SIVmac239-infected rhesus macaques. Lines represent the average percentages of cells expressing CD95 for each cell population, with error bars
representing standard errors across all 10 animals. Asterisks denote a significant difference from the day zero value based on Student’s t test (P ? 0.05).
TABLE 2. Changes in CD20?CD27?IgD?(IgM-secreting) and
CD20?CD27?IgD?(IgG/IgA-secreting) B-cell subsets
compared to baseline following SIV infection
No. of cells
No. of cells
aNumber of days postinfection with SIVmac239 (10,000 infectious units, in-
bNumber of CD20?CD27?IgD?(IgM-secreting) B cells present in periph-
eral blood at the indicated time point; values represent means of 10 animals.
cPercent change in CD20?CD27?IgD?(IgM-secreting) B cells at each time
point compared to the day zero preinfection time point.
dNumber of CD20?CD27?IgD?(IgG/IgA-secreting) B cells present in periph-
eral blood at the indicated time point; values represent means of 10 animals.
ePercent change in CD20?CD27?IgD?(IgG/IgA-secreting) B cells at each
time point compared to the day zero preinfection time point.
fInitiation of ART (PMPA and FTC).
VOL. 84, 2010 EARLY B-CELL DYSREGULATION IN SIV INFECTION2473
izing activity were comprised of clonal responses to diverse
envelope epitopes (33). In general, high-affinity antibody
(binding) did not correlate with neutralization sensitivity (func-
tionality). These data support our current study, where we
demonstrated that fewer B cells were present during the initi-
ation of the antibody response. Thus, it is likely that a small
number of B-cell clones were responsible for the antibody
response and that the breadth and potency of the response
were limited. Additionally, the paucity of memory cells during
acute infection when the initial antibody response is formed
could have led to diminished efficacies of the antibodies pro-
duced. With a reduced memory population, antibody re-
sponses have to be formed de novo, effectively resulting in each
successive SIV clone appearing as a new antigen to the anti-
body-mediated immune response. These data demonstrate
that measurement of antibody titer alone is an insufficient
means for assessment of B-cell activity and needs to be com-
bined with in-depth analysis of the B-cell compartment.
The depletion of naïve B cells from the periphery during
acute SIV infection is also likely to play a significant role in the
slow development of the B-cell response. Naïve cells dictate
the breadth and efficacy of the antibody response, requiring a
multitude of signals, including B-cell receptor activation, CD40
stimulation, and cytokine signals to initiate activation, matura-
FIG. 7. Intracellular expression of Ki67 following infection with SIVmac239. Peripheral blood from rhesus macaques infected with SIVmac239
was obtained at the indicated time points during acute and early chronic infection. Lymphocytes from whole blood analysis were analyzed for
intracellular Ki67 expression. Lines represent the average percentages of Ki67?cells within each cell population present across all 10 animals.
Asterisks denote a significant difference from the day zero time point based on Student’s t test (P ? 0.05).
FIG. 8. Measurement of SIV-specific antibody endpoint titers by concanavalin A ELISA. Envelope-specific antibody titers were measured to
concanavalin A-captured SIVsmB7 envelope proteins in longitudinal samples from rhesus macaques infected with SIVmac239. Endpoint titers are
reported as the last 2-fold dilution above the cutoff of the assay. Lines represent the log10reciprocal endpoint titers from individual animals.
Dashed lines are representative of animals with low viral setpoints (103to 106), solid lines are representative of animals with intermediate setpoints
(106to 107), and dotted lines are representative of animals with high viral setpoints (107to 108).
2474KUHRT ET AL.J. VIROL.
tion, and proliferation (25). Thus, virally induced disruption of
any of these signals could lead to decreased potency of the
antibody response. The early depletion of naïve B cells renders
the infected host more vulnerable to SIV during the initial,
critical host-pathogen interaction. Further, the failure of the
naïve population to rebound with ART suggests a limited abil-
ity for the host to recognize viral variants or new pathogens,
resulting in a diminished B-cell response to both SIV and other
opportunistic pathogens. Early antiviral treatment or thera-
peutic vaccination focused on preventing the loss of naïve B
cells during acute infection would render this critical cell pop-
ulation more effective during the chronic stage of infection.
Finally, in addition to rapid alterations in population dynam-
ics, increased activation was observed in all B-cell subsets.
Although prior studies have demonstrated increased CD95
expression on B-cell populations during chronic infections (27,
38), the precise timing of this increase was unknown. Our data
demonstrated that the increase in B cells expressing CD95
occurred almost immediately following SIV infection, was
maintained through the acute phase of infection, and was un-
affected by ART. This increased B-cell activation is of impor-
tance, as activation has also been implicated as a potential
mechanism for altered B-cell activity, i.e., poor responses to
B-cell-mediated vaccines (9). Therapeutic strategies to inhibit
chronic activation, either via CD95 or other pathways, can be
further explored using the SIV nonhuman primate model. In
contrast to activation measured by CD95 expression, prolifer-
ative responses assessed using Ki67 were variable among B-cell
subsets, indicating the potential for differential regulation be-
tween the memory and naïve B-cell subsets. Alterations in
proliferative capacity may be related to how these specific
subsets are able to respond to antigenic stimulation, and they
warrant further functional studies.
Data from the present acute study and others have demon-
strated that significant changes in total B-cell numbers occur in
the periphery following acute SIV infection. The current study
provides novel information about alterations in specific B-cell
subsets during acute SIV infection that were not revealed when
analyzing the total B-cell population. Furthermore, the data
presented in this study clearly demonstrate that measurement
of virus-specific antibody titer is not reflective of alterations in
the subset composition in the B-cell compartment. It is impor-
tant to monitor antibody specificity and functionality during
SIV infection to fully understand the extent of the damage to
the immune system. Further studies are warranted to identify
potential mechanisms for these phenotypic alterations and to
test whether concurrent functional changes in B-cell subsets
occur during the same time frame.
We thank Edmundo Kraiselburd for kindly providing the SIVsmB7
This work was funded by NIH/NIAID grants R01 AI52058 (K.S.C.)
and R01 AI35522 (D.L.S.).
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