JOURNAL OF VIROLOGY, Jan. 2009, p. 188–199
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 1
Frequency and Phenotype of Human Immunodeficiency Virus
Envelope-Specific B Cells from Patients with Broadly
Nicole A. Doria-Rose,1Rachel M. Klein,1Maura M. Manion,1Sijy O’Dell,2Adhuna Phogat,2
Bimal Chakrabarti,2Claire W. Hallahan,3Stephen A. Migueles,1Jens Wrammert,4Rafi Ahmed,4
Martha Nason,3Richard T. Wyatt,2John R. Mascola,2and Mark Connors1*
Laboratory of Immunoregulation,1Vaccine Research Center,2and Biostatistics Research Branch,3National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, and
Emory Vaccine Center, Emory University, Atlanta, Georgia 303294
Received 25 July 2008/Accepted 8 October 2008
Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic
human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that
reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of
highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural
infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and
phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various
clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were
found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term
nonprogressors. Most Env-specific antibody-secreting B cells were CD27hiCD38hiplasmablasts, and the total
plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031%
of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked
immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env
gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher
than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27?and surface IgG?.
These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B
cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for
vaccines for HIV.
There is a growing consensus that eliciting neutralizing
antibodies (NAb) will be necessary for an effective vac-
cine for human immunodeficiency virus (HIV). Historically,
many licensed vaccines have relied on the induction of
pathogen-neutralizing antibodies (reviewed in references 58
and 66). In most experimental animal model systems of viral
infection, vaccine-elicited antiviral memory T cells alone
have been unable to prevent infection or provide sterilizing
immunity (reviewed in reference 1). Similarly, in the simian
immunodeficiency virus and SHIV-nonhuman primate mod-
els of HIV, T-cell based vaccines can lower peak or setpoint
viremia after challenge but have not prevented infection (19,
76, 85). In contrast, passive antibody transfer studies in
nonhuman primate models of HIV infection have demon-
strated that neutralizing antibodies, when present at the
time of challenge, can protect from infection (43, 74, 89).
Furthermore, the risk of mother-to-child transmission of
HIV may be reduced by maternal neutralizing antibodies (9,
73). For these reasons, it is currently believed that the gen-
eration of HIV-specific NAb, possibly in combination with a
cellular immune response, should be a major goal for can-
didate vaccines for HIV.
Eliciting NAb to HIV, however, has been a challenging goal.
One of the major obstacles to the development of an effica-
cious antibody response is the extraordinary diversity of HIV
Envelope (Env) (45, 46), the surface glycoprotein that is the
target of neutralizing antibodies (70, 84). The structural fea-
tures of HIV Env, such as flexible loops and extensive glyco-
sylation, also provide resistance to neutralization (reviewed in
reference 62). To date, HIV vaccines in clinical trials have
elicited narrowly directed HIV-binding antibody with no or
weak neutralizing activity against primary isolates (30, 44, 81).
In contrast to vaccinees, most HIV-infected patients develop
neutralizing antibodies. During the early stage of infection, the
NAb response is often narrowly directed, neutralizing only
autologous isolates from earlier in the infection but not those
contemporaneous with the serum sample. It is believed that
this phenomenon is caused by progressive immunologic escape
from the neutralizing antibody response (2, 4, 53, 69, 83). In
addition, most sera from infected patients neutralize labora-
tory isolates but typically not the majority of the more difficult
to neutralize heterologous primary isolates. However, over
time some patients make broadly cross-reactive antibodies that
are able to neutralize viruses of diverse lineages, even across
clades (10, 55). Understanding the mechanism of cross-neu-
* Corresponding author. Mailing address: LIR, NIAID, National
Institutes of Health, Bldg. 10, Rm. 7N246, 10 Center Dr., Bethesda,
MD 20892. Phone: (301) 496-8057. Fax: (301) 480-9978. E-mail:
?Published ahead of print on 15 October 2008.
tralization and the generation of these antibodies may provide
information that is crucial for the development of effective
Several aspects of broadly cross-neutralizing HIV-specific
antibody responses in infected patients remain poorly under-
stood. First, the clinical characteristics of patients with a broad
NAb response remain unclear. Few studies have screened
large numbers of patients for a broad NAb response or com-
pared patients with various clinical courses. In addition, studies
have yielded variable results, using diverse assay platforms,
viral isolates, definitions of breadth, and classifications of pa-
tient clinical status (10, 17, 18, 20, 55, 63, 64). Efforts to stan-
dardize neutralization assays across laboratories have led to
the wide use of luciferase-based assays with standard panels of
pseudovirus isolates (42, 68). Several studies using this format
have examined the cross-reactivity of monoclonal antibodies
(14) and pooled sera of different clades (15), but the level of
breadth in individual patient sera has not been well described
with the current, standardized assays.
Second, the frequency and phenotype of HIV-specific B
cells in patients with broadly cross-neutralizing antibodies is
unknown. HIV?patients exhibit many altered B-cell char-
acteristics compared to uninfected donors, including in-
creased spontaneous immunoglobulin G (IgG) secretion
(37), increased expression of markers of activation and ter-
minal differentiation, and a decreased proportion of circu-
lating memory cells (reviewed in reference 50). However,
the frequency, phenotype, specificity, and immunoglobulin
class of HIV-specific B cells have not been well defined.
Enzyme-linked immunospot (ELISPOT) assays allow enu-
meration of HIV-specific antibody-secreting cells (ASC)
(27, 57, 75); however, the assay does not allow recovery of
the HIV-specific cells for later characterization or cloning.
Furthermore, to date no reagent has been available to allow
marking of individual HIV-specific cells; thus, the pheno-
typic characteristics of Env-specific B cells have remained
In the present study, we evaluated NAb breadth in a set of
113 patients and assembled cohorts of patients with or without
broadly cross-reactive NAb. Cells from these patients were
used to analyze the phenotype and frequency of Env-specific B
cells. The plasmablast subset (CD3?CD19?CD20?/loCD27hi
CD38hi) (88) was found to be increased in HIV-infected do-
nors compared to uninfected controls. In addition, the vast
majority of HIV Env-specific ASC were found within this plas-
mablast subset. Labeling Env-specific B cells permitted an ex-
amination of their frequency, immunoglobulin class, and phe-
notype. Importantly, the frequency of gp140-labeled B cells
was an order of magnitude greater than the frequency mea-
sured on the basis of ASC in ELISPOT assays. These results
describe the neutralizing antibody response in a cohort of
patients screened for broadly cross-neutralizing antibodies and
provide a characterization of peripheral blood B cells and
HIV-specific B cells of these patients. The frequency of the B
cells of these patients and the breadth of neutralization dem-
onstrate what is possible for the humoral response to HIV to
achieve and are potentially important goals for the induction of
neutralizing antibodies in vaccinees.
MATERIALS AND METHODS
Study participants. Patients in the present study signed informed consent and
participated in NIAID protocols at the National Institutes of Health, Bethesda,
MD. HIV infection was documented by HIV1/2 immunoassay. The study in-
cluded 113 HIV-infected patients who were classified as long-term nonprogres-
sors (LTNP), slow progressors, or progressors. LTNP were defined as having set
point plasma viral RNA levels of ?50 copies/ml (or below the available limit of
detection at the time of sampling), stable CD4 T-cell counts, and no opportu-
nistic infections in the absence of antiretroviral (ARV) treatment. Slow progres-
sors were defined as having a detectable viral load, a stable CD4 T-cell count
above 400 cells/?l, being diagnosed with HIV for at least 7 years, and off ARV
treatment for at least 5 years. All other patients were classified as progressors
(those off of ARV treatment for ?5 years, diagnosed with HIV-1 ?7 years prior
to sampling, and/or with CD4?T-cell counts declining and/or ?400 cells/?l). All
had been infected for at least 1 year. Some of the patients have been previously
described (40, 48). All patients were presumed to be infected with clade B virus
based on the locations of current and former residences. Phlebotomy and leu-
kapheresis were performed at time points during which patients were not using
ARVs. An additional 35 HIV-1-seronegative donors were recruited from the
National Institutes of Health donor apheresis clinic.
Storage of samples. Serum was stored at ?80°C. Peripheral blood mononu-
clear cells (PBMC) were purified from leukapheresis packs using Ficoll density
centrifugation with lymphocyte separation medium (MP Biomedicals, Solon,
Ohio). PBMC were frozen in Recovery cell culture freezing medium (Gibco,
Carlsbad, CA) using a CryoMed controlled-rate freezer (ThermoForma, Wal-
tham, MA) and stored at ?140°C. PBMC were thawed in media containing
complete RPMI with 10% fetal calf serum (FCS; Gibco) and 50 U/ml of ben-
zonase (Novagen, Darmstadt, Germany) and rested overnight in RPMI with 10%
FCS at 106cells/ml.
Flow cytometry. Multicolor flow cytometry was performed by using standard
protocols (33) using the antibodies CD3 (fluorescein isothiocyanate or Pacific
Blue), CD14 (Pacific Blue), CD19 (phycoerythrin [PE]-Cy7), CD20 (allophyco-
cyanin [APC]-Cy7), CD27 (PE), CD38 (APC), and IgG (PE-Cy5) (BD Bio-
sciences, San Jose, CA); IgA (APC) and IgM (PE) (Jackson Immunoresearch,
West Grove, PA); and Qdot605-streptavidin (Invitrogen, Carlsbad, CA). All
stainings were done at 4°C for 30 min. Plasmablasts were identified with a panel
including CD3, CD19, CD20, CD27, and CD38, with the addition of CD14
when gp140 labeling was performed (see below). The data was collected on a
FACSAria or an LSR II flow cytometer using FACSDiva software (BD
Biosciences), and cell sorting was conducted on a FACSAria. Color compen-
sation was performed using single-stained samples for each fluorochrome
used in addition to an unstained control. The data were further analyzed by
using FlowJo software (TreeStar, Cupertino, CA). A minimum of 2,000,000
events was collected for each patient sample.
Virus neutralization assays. Thawed patient serum was heat-inactivated at
56°C for 30 min prior to assay. The TZM-bl assay was used as previously
described (77). Briefly, pseudovirus stocks were prepared by transfection of 293T
cells with an env-deficient backbone (pSG3?Env) (82) and an expression plasmid
for the env gene of interest. The env genes, all of which were cloned from
CCR5-using primary isolates, were previously described (40). Serum was diluted
in Dulbecco’s modified Eagle medium–10% FCS (Gibco) and mixed with
pseudovirus. After 30 min, 10,000 TZM-bl cells were added, and the plates were
incubated for 48 h. Assays were developed with a luciferase assay system (Pro-
mega, Madison, WI), and the relative light units (RLU) were read on a lumi-
nometer (Perkin-Elmer, Waltham, MA). The percent neutralization was calcu-
lated as follows: % neutralization ? 100 ? (Vo? Vn)/Vo, where Vnis the RLU
in the virus and antibody wells and V0is the RLU in the virus-only wells. A
neutralization dose-response curve was fit by nonlinear regression using a four-
parameter hill slope equation programmed into JMP statistical software (SAS
Institute, Inc., Cary, NC). The reciprocal dilution at which 50% of the virus is
neutralized (ID50) is reported for all virus-serum pairs. Based on background
levels in sera from uninfected donors and titers against a control pseudovirus
using murine leukemia virus Env, the cutoff for neutralization was set at an
B-cell ELISPOT assay. MultiScreen-IP plates (Millipore, Billerica, MA) were
washed twice with 30% ethanol, rinsed three times with phosphate-buffered
saline (PBS), and then coated with 100 ?l of antigen in PBS at 4°C overnight. The
antigens included 2.5 ?g/ml anti-kappa light chain plus 2.5 ?g/ml anti-lambda
light chain (Rockland, Gilbertsville, PA) (anti-Ig), 10 ?g/ml HIV-1 YU2 Env
gp120 (produced in S2 cells and purified over a 17b affinity column), and 5 ?g/ml
keyhole limpet hemocyanin (Sigma, St. Louis, MO). Plates were washed six times
with PBS and blocked with RPMI with 10% FCS for 2 h at room temperature.
VOL. 83, 2009FREQUENCY AND PHENOTYPE OF HIV-SPECIFIC B CELLS189
Rested PBMC or sorted B cells were plated in at least 100 ?l of medium and
incubated overnight at 37°C. The following morning, plates were washed six
times in PBS with 0.25% Tween 20 (Sigma) (PBS-T) and incubated with 100 ?l
of 1:10,000 biotin-IgG (Jackson Immunoresearch) for 1 h at room temperature.
Plates were then washed and incubated with 100 ?l of 1:60 streptavidin-AP
(R&D Research Systems, Minneapolis, MN) for 1 h at room temperature. The
plates were then washed with PBS-T, PBS, and dI water and developed with 100
?l of BCIP/NBT (R&D Research Systems) for 20 to 30 min at room tempera-
ture, and the reaction was stopped with dI water. Spot quantitation was per-
formed by using a CTL ELISPOT reader (Cellular Technology, Ltd., Cleveland,
OH) with manual correction as needed. The average number of spots in keyhole
limpet hemocyanin wells was subtracted from the average number of spots in
anti-immunoglobulin or gp120 wells when the spots per well was calculated.
gp140 stain. Biotinylated, trimeric, cleavage-defective gp140 (biotinylated gp140-F
trimer) was used for staining patient B cells. It was derived from gp140?683(?/
FT), which consists of HIV-1 YU2 Env amino acids 1 to 683 fused to the T4
phage fibritin trimerization domain (as described in reference 91). The
gp140?683(?/FT) sequence was modified by the addition of the sequence en-
coding the Avitag signal for biotinylation (LNDIFEAQKIEWHE) at the 3? end
of the gene and subcloned into pCDNA3.1(?). The resulting plasmid was used
to transfect 293 freestyle cells at a density of 1.2 ? 106/ml using 293 fectin
(Invitrogen) according to the manufacturer’s protocol. After 4 days in culture in
shake flasks at 37°C, the supernatant containing secreted proteins was collected
by centrifugation at 3,500 rpm for 30 min. The gp140-F trimers were purified by
lentil lectin affinity chromatography, followed by chelation chromatography over
a Ni-charged column (GE Healthcare, Piscataway, NJ). Biotin ligase Bir A
(Avidity, Denver, CO) was used to biotinylate the protein at the Avitag sequence
only, distal to the relevant epitopes. Biotinylation of the gp140-F trimers was
confirmed by enzyme-linked immunosorbent assay using streptavidin-horserad-
ish peroxidase (Sigma). The antigenic structure was not affected by biotinylation,
as demonstrated by the binding of monoclonal antibodies b12, F105, and 17b
with or without soluble CD4 (unpublished data).
For staining with biotinylated gp140-F trimers, cells were thawed and rested
overnight as described above and washed in PBS with 0.5% bovine serum albu-
min (BSA; Sigma) and 0.5 mM EDTA (Quality Biologicals, Gaithersburg, MD).
A total of 50 ? 106to 150 ? 106cells in 50 ?l were blocked using unlabeled
anti-CD4 antibody (clone SK3; BD Biosciences) at 0.5 mg/ml in PBS–0.5%
BSA–0.5 mM EDTA (5) for 15 min at 4°C. Then, 8 ?g of biotinylated gp140 was
added, and the cells were incubated for 30 min at 4°C. The cells were washed
twice in PBS–0.5% BSA–0.5 mM EDTA, stained with Qdot605-streptavidin
(Invitrogen) for 30 min in the presence of cell surface antibodies at 4°C, and then
washed again. The data were collected on a FACSAria or an LSR II flow
cytometer (BD) and analyzed with FlowJo software. The Qdot605-streptavidin?
gate was positioned such that the positive population in cells stained with
Qdot605-streptavidin only (no protein) was 0.01% of the CD19?B cells. The
same gate was used for the corresponding gp140-labeled sample from the same
patient in order to exclude background staining.
Statistical analysis. The Wilcoxon two-sample test was used to compare in-
dependent groups. Paired data were compared by using the Wilcoxon signed
rank test. Correlations were determined by using the Spearman rank method.
The frequencies of not-broad neutralizing antibodies in LTNP versus all viremic
patients were compared by using the Fisher exact test. The Bonferroni method
was used to adjust P values for multiple testing.
Initial screen for broad cross-neutralizing sera. In order to
better understand the spectrum of breadth of neutralization of
HIV-1 in infected patients using current standardized assays,
we evaluated sera from a large cohort of HIV-infected patients
(n ? 113) with a variety of clinical characteristics. Table 1
shows the median and range for each patient group for CD4?
T-cell count, viral load, and number of years since HIV diag-
nosis at the time of screening. Twenty-four patients were
LTNP, also called elite controllers, from the cohort described
previously (47, 48); they control viremia without the use of
ARVs to ?50 copies/ml of plasma, with a median CD4?T-cell
count of 964 cells/?l. Thirty-seven patients were classified as
slow progressors (defined as having a stable CD4?T-cell count
of ?400 cells/?l, no ARV within 5 years, and infected ?7
years). The remaining 52 patients were classified as progres-
sors (those patients with ?400 CD4?T cells or declining,
and/or having taken ARVs within 5 years of screening, and/or
diagnosed 1 to 7 years prior to screening). All patients were off
of ARV at the time of screening.
For the purpose of screening for breadth of neutralization,
we used a TZM-bl cell line-based HIV neutralization assay (42,
77) with panels of isolates from clades A, B, and C. All assays
used pseudoviruses made with primary isolate env genes. The
initial screen of 113 sera was performed with a five-isolate
minipanel, and the findings were validated for a subset of sera
(n ? 42) with an extended panel of 20 isolates from clades A,
B, and C (see below). The number of isolates neutralized in the
set of five correlated well with the number neutralized in the
set of 20 (r ? 0.84, P ? 0.001). The minipanel consisted of
JRFL (clade B), CAAN.A2 (clade B), THRO.18 (clade B),
Q168.a2 (clade A), and ZM106.9 (clade C). In a previous
analysis with a subset of these sera, JRFL and Q168.a2 were
moderately sensitive, CAAN.A2 and THRO.18 were moder-
ately resistant, and ZM106.9 was highly resistant to neutraliza-
tion by patient sera (40).
Using the minipanel, we found a range of neutralization
breadth among the 113 sera. As shown in Fig. 1A, 38% of sera
were able to neutralize four or five of five isolates and were
classified as broad; 33% of sera were able to neutralize two or
three of five isolates and were classified as intermediate; and
29% of sera neutralized zero or one of five isolates and were
classified as not broad. Broad sera were found in all patient
groups (Fig. 1B). There was less breadth in the LTNP group,
with 50% of the LTNP sera classified as not broad compared to
27 and 21% in slow progressors and progressors, respectively
(P ? 0.04, Fisher exact test). Conversely, only 25% of the
LTNP sera were classified as broad compared to ?40% in each
of the viremic groups.
We then sought to further extend the characterization of the
neutralizing antibody response. Using the expanded panel of
20 isolates, we determined the serum titers from a subset of
patients that had been characterized as broad or not broad in
the initial screen. Isolates from clades A, B, and C with a range
of neutralization sensitivities (38–40) were included. Figure 2
shows the ID50neutralization titers for the expanded panel,
along with the clinical characteristics of 22 patients. Nine of the
sera were remarkably broad, neutralizing fifteen or more iso-
lates, including most of the clade A and C viruses. Patients in
the broad group had higher plasma viral RNA compared to
those in the not-broad group (median, 11,650 versus 1,289
RNA copies/ml, P ? 0.003), while the CD4 count and years
TABLE 1. Clinical characteristics of patient groups
Viral RNA copies/
No. of CD4?
No. of yr
190 DORIA-ROSE ET AL.J. VIROL.
since diagnosis with HIV did not correlate with breadth (P ?
0.2 and P ? 0.5, respectively).
We next characterized the frequency and phenotype of B cells
in the 13 patients with broad sera, 9 with not-broad sera, 8 addi-
tional HIV-infected patients, and 18 uninfected donors. Patient
sera were classified as broad or not broad based on the five-virus
minipanel. When the cells of all 30 patients were examined, the
frequency of B cells in lymphocytes was lower in patients than in
uninfected controls, with median frequencies of 6.0 and 15.8%,
respectively (P ? 0.001). No significant differences emerged be-
tween patients with or without broad NAb in the frequency of B
cells or IgG?B cells (data not shown). The levels of CD19?
CD27?memory B cells were somewhat lower in the patients with
broad sera relative to not broad sera (median 20% of B cells
versus 35%), although this trend did not reach statistical signifi-
cance. CD19?CD27?frequency showed a weak but significant
negative correlation with viral load (r ? ?0.49, P ? 0.02) when all
patients were analyzed.
HIV-specific ASC. In order to examine the phenotype of
HIV-specific B cells, we first searched for B-cell subsets that
are enriched for cells actively secreting Env-specific IgG. We
hypothesized that plasmablasts would be such a population,
since they have been shown to represent most of the ASC in
the peripheral blood of uninfected donors and are increased in
frequency during the acute response to vaccination (60, 88).
We therefore examined whether the plasmablast subset is also
increased in chronic HIV infection. Figure 3A shows typical
staining for plasmablasts, defined as: CD3?CD19?CD20?/lo
CD27hiCD38hi(60, 88). We found that HIV-infected patients
had a significant increase in plasmablast frequency compared
to uninfected donors, with medians of 1.6 and 0.46% of B cells,
respectively (P ? 0.001) (Fig. 3B). The fraction of plasmablasts
that were surface IgG?was also higher in HIV-infected pa-
tients (median of 32.3% versus 21.3%, P ? 0.04). The plasma-
blast frequency was not different between the broad and not-
broad groups and did not correlate with viral load or CD4?
T-cell count (data not shown).
FIG. 1. Distribution of broadly cross-reactive sera. (A) Percentages of
113 sera that neutralize the indicated number of isolates. Sera that neutralize
none or one of five are classified as not broad (blue), two or three of five as
intermediate (white), and four or five of five as broad (green). (B) Percent-
ages of broad, intermediate, and not-broad sera in LTNP, slow progressor,
and progressor patient groups (colors are as defined in panel A).
FIG. 2. Clinical characteristics and neutralization data for patients in B-cell analysis. Each column shows data from a single patient. In the top panel,
each row shows the ID50of serum against the indicated virus isolate. ID50values: white, ?100; yellow, 100 to 400; orange, 401 to 1,000; red, ?1,000. In
the bottom panel, abbreviations are defined as follows: VL, viral load in copies/ml; CD4, CD4?T cells/?l; Yrs Since Dx, years since HIV diagnosis.
Clinical data are from the same time point or within 3 months of the serum sample. Broad and not-broad categories are shown as classified for Fig. 1.
VOL. 83, 2009FREQUENCY AND PHENOTYPE OF HIV-SPECIFIC B CELLS 191
Using a B-cell ELISPOT assay, we measured the cells that
actively secrete IgG or HIV Env-specific IgG. HIV gp120 was
used as the target antigen on ELISPOT plates; trimeric gp140
gave equivalent results to gp120 (data not shown). One might
expect to find differences in the responses to these two proteins
since a gp120 ELISPOT assay does not detect cells specific for
gp41 and an ELISPOT assay using intact gp140 trimer is un-
able to detect gp120 epitopes that are buried in the trimer. It
is possible that increases in detection of gp41-specific cells with
the gp140 protein are offset by the loss of inner binding sites on
gp120. Alternatively, the fraction of the total envelope re-
sponse comprised by these specificities is below the limit of
detection in our assay. In this assay, 0.07% of unstimulated
PBMC secreted IgG (Table 2). The overall frequency of
gp120-specific IgG ASC varied considerably between patients,
with a median of 2.5 per 106PBMC (range, 0 to 17 per 106, n ?
15), a finding similar to the range reported by other groups (57,
75). We next purified B cells and B-cell subpopulations by
fluorescence-activated cell sorting and evaluated the sorted
cells by ELISPOT assay. A total of 0.0031% of B cells secreted
anti-gp120 IgG. Cells secreting anti-gp120 IgG were largely
limited to the plasmablast population (Fig. 4): a median of
58% of IgG ASC and up to 92% of the gp120-specific ASC
were plasmablasts. The median frequency of anti-gp120 IgG
ASC in 11 patients was 0.047% of total plasmablasts and
0.49% of IgG-secreting plasmablasts (Table 2). The frequency
of gp120-specific IgG ASC in PBMC or in plasmablasts did not
correlate with viral load, CD4 count, or years since diagnosis at
the time of sampling. Likewise, no differences were noted
between broad and not-broad cohorts for the frequencies
We were unable to immortalize sorted plasmablasts by in-
fection with Epstein-Barr virus (EBV). Seven attempts using a
high-titer EBV stock to infect plasmablasts yielded no trans-
formants, whereas nonplasmablast, CD20?/loB cells from the
same samples were consistently transformed (data not shown).
This was not due to cell death during sorting, since the sorted
plasmablasts survived to secrete IgG in the ELISPOT assay.
Nor was it a problem with receptor expression, since most
plasmablasts expressed CD21, the main receptor for EBV
(data not shown).
Frequency and phenotype of envelope-specific B cells. In
order to directly analyze the frequency and phenotype of HIV-
specific B cells by flow cytometry, we developed a protocol for
staining B cells with a recombinant HIV Env protein, biotin-
ylated gp140-F trimer (hereafter referred to as gp140). This
method allows the measurement of total Env-specific cells.
Trimeric gp140 was chosen because it better reflects the con-
formation and epitopes of the native Env spike than mono-
meric gp120 (90, 91). Although the ELISPOT assay, described
above, measures the cells that are actively secreting anti-Env
IgG (mainly plasmablasts), it does not account for the large
fraction of peripheral antigen-experienced B cells that are not
actively secreting antibody. The ELISPOT assay therefore was
likely to underestimate the frequency of total Env-specific B
cells. Cells were stained with biotinylated gp140 and detected
with streptavidin-Qdot605. Figure 5 shows representative ex-
amples of staining CD19?cells with gp140. To validate the
specificity of staining, we compared gp140-labeled B cells in
HIV-infected patients to labeled cells from uninfected donors.
Background levels were assessed on cells stained with strepta-
vidin-Qdot605 only. The Qdot605-Streptavidin?gate was po-
sitioned such that the positive population in cells stained with
Qdot605-streptavidin only (no protein) was 0.01% of the
CD19?B cells. The same gate was used for the corresponding
gp140-labeled sample from the same patient. The level of
binding to B cells of HIV-uninfected controls was similar to the
background due to streptavidin-Qdot605 alone (median of
0.021% [range, 0.008 to 0.029%] in eight donors). B cells
labeled with gp140 were clearly visible in HIV-infected pa-
tients (Fig. 5A). The median frequency of gp140 binding to B
cells of 14 HIV-infected patients was 0.09% (range, 0.028 to
0.21%) (Fig. 5B). The frequency of gp140-labeled B cells in
individual patients did not correlate with breadth of NAb, viral
load, CD4 count, or years since HIV diagnosis at the time of
sampling. Since the recombinant Env proteins used in both the
ELISPOT assay and B-cell staining were derived from HIV-1
strain YU2, we compared neutralization titers against HIV-1
YU2 to data from both assays and did not find significant
correlations (data not shown).
We then examined the phenotype of Env gp140-specific
peripheral B cells and compared the results to total B cells
FIG. 3. The plasmablast frequency is increased in HIV-infected patients. (A) Flow cytometric analysis of plasmablasts. A representative sample
is shown. Plasmablasts are CD3?CD19?CD20?/loCD27hiCD38hi. The large gate in the forward-scatter/side-scatter plot was set to include
plasmablasts based on backgating. (B) Plasmablasts as a percentage of CD19?lymphocytes in HIV-positive patients and uninfected donors.
Horizontal bar, median value.
TABLE 2. Frequencies of ASC in different cell populations
% IgG secretinga
% gp120 specificb
as % of IgG
aPercentage of input cells secreting IgG in an ELISPOT assay.
bPercentage of input cells secreting anti-gp120 IgG.
cFrequency of gp120-specific/frequency of IgG secreting ? 100.
192 DORIA-ROSE ET AL. J. VIROL.
(Fig. 6A to C). Surface expression of the activation marker
CD38 was highly variable between patients and, overall,
CD38intermediateand CD38hicells were present among gp140-
labeled cells at frequencies close to those of total B cells in
each patient. A total of 64% of gp140-labeled B cells expressed
the memory marker CD27 compared to 25% of all CD19?B
cells. Conversely, the frequency of gp140-labeled cells was
0.17% (range, 0.08 to 0.41%) in the CD19?CD27?cells,
higher than in total B cells (0.09%). Although it has been
shown in humans that all CD19?CD27?cells are memory
cells, not all memory B cells are CD27?(25, 26). We therefore
used surface immunoglobulin class as an indicator of switched
memory phenotype (Fig. 6D). gp140-labeled B cells were
highly enriched for surface IgG compared to total B cells, with
a median 9.7% of total B cells but 48% of gp140-labeled B cells
expressing surface IgG, and a concomitant reduction of surface
IgM?cells (medians of 81% versus 25%, respectively). The
surface IgA frequency was 6.0% in the gp140-labeled cells, a
level similar to that in total B cells. Together, these data indi-
cate that the majority of gp140-labeled B cells were class-
switched memory cells. Total gp140-labeled B cells were 50-
fold more frequent and IgG?gp140?B cells were 15-fold more
frequent than gp120-specific ASC within B cells of the same
patients, indicating that the ELISPOT assay underestimates
the true frequency of Env-specific B cells. The frequency of
gp140-specific plasmablasts was also examined (Fig. 6E). A
median of 1.8% of gp140-labeled B cells were plasmablasts,
similar to total B cells. A total of 0.05% of plasma-
blasts stained positive for gp140, while 0.07% of the plas-
mablasts from the same patients secreted anti-Env gp120
antibody in the ELISPOT assay. Thus, although the ELISPOT
assay underestimated the frequency of total HIV-specific B cells,
the numbers of Env-specific cells within the plasmablasts were
concordant when measured by gp140 staining or by ELISPOT
The study of broadly cross-reactive NAb and the cells that
make them is a critical line of research for HIV vaccine devel-
opment. In the present study, we assembled cohorts of patients
with or without broadly cross-reactive neutralizing antibodies
and explored the frequency and phenotype of Env-specific B
cells in these patients. The prevalence of patients with broadly
cross-neutralizing sera was 38%. In addition, several sera
showed extraordinary breadth, neutralizing most primary iso-
lates studied from clades A, B, and C. Investigations of the B
cells from these patients showed that the HIV Env-specific
ASC frequency was not different between patients with or
without broad NAb and that the plasmablast B-cell subset
contained the majority of active Env-specific ASC. We have
also developed a staining technique that, for the first time,
allows flow cytometric analysis of individual Env-specific B
cells. Measurements of the total Env-specific B-cell population
showed that their frequency was 50-fold larger than estimates
based upon ASC in ELISPOT assays.
The prevalence of patients with broadly cross-neutralizing
antibodies was greater than that observed in prior studies. A
total of 42 of the 113 patient sera in the present study could
neutralize four or five out of five isolates, and ?70% could
neutralize two or more. An extended analysis (Fig. 2) showed
that nine extremely broad sera were able to neutralize at least
15 of 20 isolates, including most of the neutralization-resistant
and non-clade B viruses. In the present study, a broadly cross-
neutralizing antibody response was much less common among
LTNP (elite controllers) than viremic patients, a finding that is
consistent with other recent observations (8, 63). We also
found that the progressor and slow progressor groups did not
differ in the proportion of patients with breadth (Fig. 1B),
which is similar to recent findings in a different cohort (63).
Earlier studies, using different assays and patient group defi-
FIG. 4. gp120-specific ASC are plasmablasts. The top panels present representative flow cytometry data showing gating strategy for plasma-
blasts. The bottom panels show representative anti-gp120 IgG ELISPOT wells from the indicated populations. Input cell numbers: CD19?well,
30,000; CD19?CD20?well, 200,000; CD27?/loCD38?/lo, 25,000; plasmablasts, 4,000.
VOL. 83, 2009FREQUENCY AND PHENOTYPE OF HIV-SPECIFIC B CELLS193
nitions, found more broad NAb in the sera of slow progressors
(16, 18, 20, 52, 64, 93), although these comparisons were made
to patients with severe CD4?T-cell depletion and a global
decline in immunity. Some previous studies using a PBMC-
based assay with multiple rounds of viral replication have
found at least some cross-neutralization of primary isolates
within clade B in more than half of chronically infected pa-
tients (18, 53), but others found a much more limited preva-
lence of breadth when isolates of non-B clades were included
(11, 55). The high proportion of sera able to neutralize multi-
ple heterologous and non-B primary isolates in the present
study implies that breadth is more common than previously
thought. It remains possible that this finding is due to differ-
ences between the PBMC-based assay and the cell-line based
TZM-bl assay. Indeed, discordant results have been reported
using the same virus-serum pairs in both assays (14), although
the TZM-bl assay does not always give higher titers (68). Ul-
timately, the relevance of the neutralization breadth and titer
measured in this assay can only be confirmed by in vivo studies
of passive transfer and vaccine efficacy trials. However, if the
same assay platform is used in vaccine clinical trials, then the
data presented here set a standard to which vaccine samples
can be compared.
These results confirm and extend prior work that indicated
that it is possible for the human B-cell response to generate
cross-neutralizing antibodies and potentially set a goal for im-
munization. Early candidate vaccines focused on eliciting an-
tibodies using recombinant Env proteins, but the antibodies
made by vaccinees had neutralizing activity only against labo-
ratory strains, not primary isolates (29, 44), and the vaccines
had no protective efficacy (65). A recent phase I study of DNA
prime, recombinant Env gp120 boost regimens found weak
neutralizing antibody with modest breadth (81); the titers were
an improvement over other previous studies, but much less
than the levels we found in most patients using the same assays.
By measuring the levels and the breadth of NAb in patients, we
describe what is possible for the immune system to achieve,
and these results may provide some reference for the level of
breadth and titer that is potentially achievable in vaccinees.
The development of broadly cross-neutralizing antibodies
may be partially dependent on ongoing exposure of B cells to
HIV antigen. Half of the aviremic LTNP had sera classified as
not broad, neutralizing only one or none of the five test iso-
lates. In other cohorts of LTNP, autologous neutralizing activ-
ity was found to be very low (7) and heterologous neutraliza-
tion was rare (63). Among the viremic patients studied here,
viral load had a weak, positive association with breadth (P ?
0.003), similar to some previous observations (23). ARV treat-
ment can lead to declines in both neutralizing activity (7) and
HIV-specific ASC (57). Collectively, these findings support a
link between prolonged exposure to viral antigen and the de-
velopment of cross-reactive NAb. However, antigen exposure
is not always predictive of breadth, since some patients failed
to develop broad NAb even after 20 years of untreated viremia
(Fig. 2), while some aviremic LTNP did develop broad NAb
(Fig. 1B). Thus, viremia is likely an important but not exclusive
driving force in the development of broad NAb.
We also explored the frequency and phenotype of ASC in
patients with or without broadly cross-neutralizing antibodies.
A population of particular interest was CD19?CD20?/lo
CD27hiCD38hiplasmablasts, which are derived from memory
B cells, as shown by their extensively mutated immunoglobulin
genes (88). Upon activation by antigen or within days of vac-
cination, memory B cells proliferate and differentiate into plas-
mablasts, which appear at high frequencies and are the major
ASC in the peripheral blood (60, 88). Most of these cells die by
apoptosis, while some traffic to bone marrow or inflamed tissue
and become long-lived plasma cells that maintain continuous
production of antibodies (reviewed in references 32 and 87).
Plasmablasts are also increased during episodes of active sys-
temic lupus erythematosus (59), infectious mononucleosis
(49), and acute rotavirus infection (34) and appear at a higher
frequency in the cerebrospinal fluid of HIV-infected patients
compared to controls (21). We show here that in the setting of
chronic HIV infection, plasmablast frequency in the blood is
significantly higher in patients than in uninfected donors (1.6%
versus 0.46%, P ? 0.001). In addition, plasmablasts accounted
for most of the ASC in HIV?patient PBMC. Although Env-
FIG. 5. gp140 specifically stains Env-specific B cells. (A) PBMC
were labeled with biotinylated gp140-F trimer (right panels) or buffer
only (left panels) and later stained with Qdot605-streptavidin, along
with antibodies for lymphocyte markers. Panels show CD3?CD14?
CD19?cells. The gate shows gp140-labeled cells and is set so that
?0.01% of cells are positive in the streptavidin-only sample for each
patient. Representative plots of an uninfected control (top) and an
HIV-infected patient (bottom) are shown. (B) Percent of B cells that
are labeled with gp140?in HIV-infected patients and uninfected
194DORIA-ROSE ET AL.J. VIROL.
FIG. 6. Frequencies of B cells and gp140-labeled B cells expressing surface markers. (A) Left panel, CD3?CD19?cells from a representative
patient. Right panels, CD3?CD19?cells of the same sample stained for B-cell markers. gp140-labeled cells (blue) are shown on the background
of all B cells (red). Totals of 1.8 ? 105to 3.4 ? 105gated events are shown. (B) Gating strategy for CD27 and CD38. Left panel, CD3?CD19?
cells from a representative patient. Right panel, gp140-labeled cells (blue) on the background of total B cells (red). (C and D) Percent of cells of
the indicated phenotype in total B cells or gp140-labeled B cells. Horizontal bars, medians. (E) Left panel, CD3?CD19?cells from a
representative patient. Center panel, CD3?CD14?CD19?CD20?/locells of the same sample. The CD27hiCD38hicells are plasmablasts.
gp140-labeled cells (blue) are shown on the background of other B cells (red). A total of 4.7 ? 105gated events are shown. Right panel, percentage
of B cells or gp140 labeled B cells that are plasmablasts.
VOL. 83, 2009 FREQUENCY AND PHENOTYPE OF HIV-SPECIFIC B CELLS195
specific ASC were found to be almost entirely contained within
this population, there was no relationship between the fre-
quency of ASC or Env-specific plasmablasts and the develop-
ment of a broadly cross-neutralizing antibody response. Al-
though viral load had a positive association with NAb breadth,
CD4?T-cell counts, years since diagnosis, and the other B-cell
subsets examined did not correlate with broad NAb in these
The observation that the majority of cells actively secreting
anti-Env antibodies are plasmablasts has implications for the
generation of Env-specific monoclonal antibodies. Since the
cells that actively secrete anti-Env gp120 IgG are plasmablasts,
we attempted to generate immortalized cell lines from sorted
plasmablasts by EBV transformation but were unable to do so
in seven attempts. Prior work has suggested that this difficulty
is caused by lytic infection of highly activated B cells. Immor-
talization of B cells by EBV requires latency, as opposed to
lytic replication. The EBV gene BZLF-1 switches viral repli-
cation to the lytic phase, and there is both direct and indirect
evidence for its upregulation in plasmablasts. Plasmablasts ex-
press XBP-1 (6), a B-cell transcription factor that transacti-
vates BZLF-1 (12, 79), and it has been directly shown that
BZLF-1 is upregulated in CD38hiB cells (36). EBV transfor-
mation alone will not capture the ASC, such as plasmablasts,
that may contain the specificities contributing to neutraliza-
tion. This may explain difficulties encountered by many labo-
ratories in cloning neutralizing antibodies against HIV via
EBV immortalization. Single-cell PCR and cloning of B-cell
antigen receptors of sorted plasmablasts (88) or gp140-stained
cells allows the immortalization step to be bypassed and thus
may be a viable alternative method for obtaining monoclonal
antibodies from these cells.
Use of a technique that permits direct staining of Env-specific
B cells permitted an investigation of their frequency and pheno-
type. Cells actively secreting antibody make up only a subset of all
antigen-experienced B cells, and therefore ELISPOT assays are
likely to underestimate the frequency of total Env-specific B cells.
Furthermore, since cells are destroyed during the assay, we could
later characterization or cloning. For these reasons, we devel-
oped techniques to measure the total frequency of Env-specific
B cells using direct staining by a labeled Env protein, biotin-
ylated gp140-F trimer. We observed that gp140-labeled B cells
are mainly but not entirely CD27?and half express surface
IgG. The proportion that express IgM was low but variable.
The majority of these cells are most likely antigen-experi-
enced cells, given that they were not found above the limit
of detection in uninfected controls and are consistent with
the large fraction of human memory cells that are IgM?(3,
80). Unexpectedly, the frequencies of CD38intermediateand
CD38hiB cells and of plasmablasts were not different in
gp140-labeled cells compared to total B cells, suggesting
that Env-specific cells are not more activated than those of
other specificities during chronic viremia. In addition, no
differences were found in these surface phenotypes between
patients with or without broad NAb. However, it should be
noted that these surface markers may not reliably discrim-
inate functional memory or activated B cells. CD27?mem-
ory B cells have been described in normal donors (25, 26,
86), and a population of CD27?memory B cells that are
specific for HIV in an ELISPOT assay was recently de-
scribed (51). Likewise, the expression of the activation
marker CD38 can be highly heterogeneous (71). The use of
these markers to distinguish B-cell activation and memory
subsets is still being refined (71, 80).
The frequency of B cells staining with Env gp140 was con-
siderably higher than the frequency of Env-specific ASC ob-
served. The ELISPOT assay, which did not include an in vitro
activation step, only measured cells that were actively secreting
antibody, and not all HIV-specific cells would be expected to
be ASC at any given time. Thus, the ELISPOT data greatly
underestimated the number of Env-specific B cells. This was
not simply due to differences in the techniques of measure-
ment. Previous studies using an ELISPOT assay to measure
HIV-specific B cells in unstimulated PBMC found frequencies
similar to those reported here (57, 75). In vitro stimulation of
B cells might be expected to allow measurement of more of the
HIV-specific cells, but in one study using this approach the
frequency within B cells of Env-specific ASC was similar to our
findings for unstimulated cells (27). In addition, although mo-
nomeric gp120 was used for ELISPOT assay while trimeric
gp140 was used for staining, this difference did not influence
the results as the two proteins gave equivalent data when used
side-by-side in ELISPOT assays. Therefore, we infer that tech-
nical issues do not account for this difference. However, within
the plasmablast subset, the frequencies of Env-specific stained
cells and ASC were very similar (medians of 0.05% versus
0.07%). Thus, for the subpopulation that is most likely to
secrete antibody, the ELISPOT and staining assays did give
similar results. Therefore, these results suggest that the vast
majority of HIV-specific B cells are not ASC and not measured
in the ELISPOT assay and that the true frequency of
HIV-specific B cells is considerably higher than previously appre-
Several studies that compare the antigenic structures of re-
combinant Env proteins suggest that trimeric gp140 is a better
mimic of the conformation of native gp160 spike than gp120
monomer (13, 61, 78, 90, 91). Investigations of the trimeric
gp140-F construct used here (91) showed better binding of
neutralizing MAb, including b12, to trimeric gp140 than to
gp120. Conversely, the trimeric gp140 was bound less well by
non-neutralizing MAb that recognize epitopes thought to be
buried in the trimeric spike structure, such as C11 (54), imply-
ing exposure of relevant epitopes and occlusion of non-neu-
tralizing epitopes (91). In addition, there is some evidence that
trimeric gp140 elicits more and/or broader neutralizing anti-
bodies than gp120 when used as an immunogen (24, 31, 35, 41,
92). However, the gp140-F trimers are not perfect mimics
of the gp160 spike: they contain non-HIV protein domains,
and the cleavage site between gp120 and gp41 is mutated in
order to stabilize the quaternary structure. These factors, in
addition to the reduced binding to non-neutralizing antibodies,
suggest that the frequencies of Env-specific B cells reported
here may still be an underestimate.
It remains unknown whether patients with broadly cross-
reactive neutralizing activity have an oligoclonal response, tar-
geting one or a few highly conserved epitopes via antibodies
akin to the known monoclonal antibodies or whether they
produce a variety of antibody species, each targeting epitopes
found on a subset of isolates. The latter possibility seems less
196DORIA-ROSE ET AL. J. VIROL.
likely, given that neutralization in many broad patients ex-
tended to viruses in clades A and C that represent envelopes to
which they were unlikely to be exposed. Rather, it is more
likely that this breadth is mediated by a limited number of
clones directed against conserved epitopes. An early analysis of
two of the broad-serum patients within this cohort supports
this hypothesis. The majority of neutralization was attributable
to anti-gp120 antibodies with limited specificities. In one pa-
tient this was almost exclusively mediated by antibodies di-
rected to the CD4 binding site, while for the second patient
there was substantial anti-CD4 binding site activity, as well as
activity directed to additional non-V3 epitopes on gp120 (40).
Phage display and panning technology have been used to gen-
erate monoclonal antibodies from patients with broadly cross-
neutralizing antibodies; studies of one patient have yielded
three broadly cross-neutralizing monoclonal antibodies against
gp120 and gp41 (22). Thus far, the full range of specificities
that mediate the breadth of polyclonal sera in a single individ-
ual have not been determined at the monoclonal antibody
level, in part because of the extremely low frequencies of
neutralizing antibodies in vivo. Enrichment of Env-specific B
cells using the techniques described here and sorting for plas-
mablasts or for Env-specific B cells may be helpful in achieving
In summary, the results of the present study provide a con-
text for the induction of humoral immunity by HIV vaccines.
The frequency and phenotype of HIV-specific B cells reported
here, although not correlated with breadth, are examples of
what is potentially achievable by the human immune response.
In addition, the number of isolates neutralized and the titer of
neutralizing antibodies are goals that are theoretically achiev-
able in prophylactic vaccines. Further work, using the direct
staining techniques described here for isolation of HIV-spe-
cific B cells, could potentially uncover the specificities and
mechanism by which some patients mediate broad cross-neu-
We thank James Arthos, Jeffrey Cohen, Jason Ho, Laurie Lamor-
eaux, and Mark Louder for technical assistance and invaluable advice
and Nancy Cogliano-Schutta, Catherine Rehm, Gregg Roby, and Sara
Stallings for coordinating samples and patient visits.
This research was supported by the Intramural Research Program of
the National Institute of Allergy and Infectious Diseases, National
Institutes of Health, and a grant from the NIH Intramural AIDS
Targeted Antiviral Program.
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VOL. 83, 2009 FREQUENCY AND PHENOTYPE OF HIV-SPECIFIC B CELLS199