Th2 Polarization in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus (HIV)-Infected Subjects, as Activated by HIV Virus-Like Particles

Abstract

We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14(+) cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14(+) circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of "responsive" vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.

Full text

JOURNAL OF VIROLOGY, Jan. 2009, p. 304–313 Vol. 83, No. 1
0022-538X/09/$08.00⫹0 doi:10.1128/JVI.01606-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Th2 Polarization in Peripheral Blood Mononuclear Cells from Human
Immunodeficiency Virus (HIV)-Infected Subjects, as
Activated by HIV Virus-Like Particles
䌤
L. Buonaguro,
1,2
M. L. Tornesello,
1
R. C. Gallo,
2
Franco M. Marincola,
3
G. K. Lewis,
2
and F. M. Buonaguro
1
*
Laboratory of Molecular Biology and Viral Oncogenesis & AIDS Reference Center, Istituto Nazionale Tumori Fond. G. Pascale, Naples, Italy
1
;
Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
2
; and Infectious Disease and
Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center, National Institutes of
Health, Bethesda, Maryland
3
Received 29 July 2008/Accepted 10 October 2008
We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55
gag
virus-like particles
(HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan
HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with
a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a
primary and secondary response in autologous human CD4
ⴙ
T cells in an ex vivo immunization assay. In the
present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells
(PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the
immune activation status and the responsiveness of circulating monocyte CD14
ⴙ
cell populations to an
immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation
induced by HIV-VLPs in CD14
ⴙ
circulating cells, which does not consistently correlate with an advanced status
of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by
HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants
would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility
of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the
identification of “responsive” vaccinees as well as the understanding of eventual failures in individuals enrolled
in clinical trials.
Virus-like particles (VLPs) represent a form of subunit vac-
cine based on viral capsid proteins which self-assemble into
particulate structures closely resembling immature virus parti-
cles (18, 28, 33, 40). VLPs are replication and infection incom-
petent, lacking regulatory proteins and infectious genetic ma-
terial, and can be employed to deliver antigenic structures. In
particular, VLPs also efficiently reach the major histocompat-
ibility complex class I pathway in antigen-presenting cells
(APCs) in the absence of infection or intracellular replication
(3, 49, 50). Considering these properties, VLPs represent a
highly attractive vaccine approach and have been produced
from a broad spectrum of enveloped and nonenveloped vi-
ruses, regardless of whether the particle structure is based on
single or multiple capsid proteins (42).
The VLPs developed in our laboratory are based on the
human immunodeficiency virus type 1 (HIV-1) Pr55
gag
precur-
sor protein (HIV-VLPs) and display an entire gp120 molecule
from a Ugandan HIV-1 isolate of the A clade (HIV-VLP
A
s)
(6, 8), anchored through the transmembrane portion of Ep-
stein-Barr virus gp220/350 (7). These HIV-VLP
A
s induce
HIV-1-specific CD4
⫹
and CD8
⫹
T-cell responses and cross-
clade neutralizing antibodies in immunized BALB/c mice (11).
Moreover, the intraperitoneal and intranasal administration of
HIV-VLP
A
s in mice induces antibody responses at systemic
and mucosal (vaginal and intestinal) levels (9, 13).
Dendritic cells (DCs) are professional APCs specialized to
capture and process antigens in vivo (30), converting proteins
to peptides that are presented on major histocompatibility
complex molecules and recognized by T cells. DCs also migrate
to T-cell areas of lymphoid organs, where the two cell types
interact to bring about clonal selection (32, 47, 55). In partic-
ular, two main DC types are present in human peripheral
blood, known as myeloid DCs, the major subset, representing
around 80% of blood DCs (23), and plasmacytoid DCs.
DCs derive from circulating monocytes which are character-
ized by expression of large amounts of CD14. However, it has
been shown that monocytes in human peripheral blood are
heterogeneous in terms of expression of antigenic markers
(namely, CD14 and CD16), correlating to possible differential
physiological activities of monocyte subsets: in particular,
CD14
hi
CD16
⫺
cells, which are often called classic monocytes,
and CD14
⫹
CD16
⫹
cells, which resemble mature tissue mac-
rophages and appear more likely to be precursors of DCs (43,
48, 56). Nevertheless, both cellular subsets can differentiate ex
vivo into DCs in the presence of granulocyte/macrophage col-
ony-stimulating factor and interleukin-4 (IL-4) (51, 52).
* Corresponding author. Mailing address: Laboratory of Molecular
Biology and Viral Oncogenesis & AIDS Reference Center, Istituto
Nazionale Tumori Fond. G. Pascale, Via Mariano Semmola, 1, 80131
Naples, Italy. Phone: 39-81-5903.830. Fax: 39-081-5451276. E-mail:
irccsvir@unina.it.
䌤
Published ahead of print on 22 October 2008.
304

An additional monocyte subset, defined by the expression of
CD14, CD16, and CD64, has lately been reported, combining
characteristics of monocytes and DCs, with high expression of
CD86 and HLA-DR and high T-cell-stimulatory activity (29).
We have recently shown that baculovirus-expressed HIV-
VLPs are able to induce maturation and activation of mono-
cyte-derived DCs (MDDCs) and that this effect is partially
mediated by the internal Toll-like receptors 3 and 9 (12). The
HIV-VLP-activated MDDCs produce a pattern of cytokines
indicative of both Th1 and Th2 pathways and induce primary
and secondary responses in autologous human CD4
⫹
T cells in
an ex vivo immunization assay. The uptake of HIV-VLPs by
DCs appears to be mainly mediated by a cytochalasin-D-sen-
sitive pathway (12). Moreover, the HIV-VLP-activated MD-
DCs show specific transcriptional profiles of genes involved in
the morphological and functional changes characterizing
MDDC activation and maturation (1).
In addition, we have shown that the morphological and gene
transcriptional maturation pattern induced by HIV-VLPs in ex
vivo-generated MDDCs can also be observed in CD14
⫹
un-
cultured peripheral blood mononuclear cells (PBMCs) (10).
This experimental approach would greatly facilitate screening
for responsiveness to vaccines and an understanding of even-
tual failures in individuals enrolled in clinical trials.
In an HIV-1 vaccine perspective, and in particular for
therapeutic strategies, it is mandatory to evaluate the impact
of HIV seropositivity status on responsiveness to immuni-
zation, considering a possible quantitative and/or qualitative
impairment in circulating monocytes, DCs, and other APCs
(4, 21, 22, 38).
In this study, the response to HIV-VLPs has been evaluated
with CD14
⫹
circulating monocytes, without distinction be-
tween different CD16 (⫹or ⫺) subsets, according to HIV-1
seropositivity status. An informative evaluation of PBMCs
showing the individual vaccinees’ immune responsiveness to a
human papillomavirus-VLP vaccine has been reported (27, 45,
46). The results here reported indicate that the HIV-1 sero-
positivity status, with either low or high viremia, does not
impair the immune activation status and the responsiveness of
circulating monocyte CD14
⫹
-cell populations to an antigenic
stimulus. Nevertheless, a complete lack of maturation induced
by HIV-VLPs in CD14
⫹
circulating cells is occasionally ob-
served for a few HIV-1-seropositive subjects, without any con-
sistent correlation to an advanced status of HIV-1 infection
(high viremia levels and/or CD4
⫹
counts). However, the es-
tablished Th2 polarization in both HIV-seropositive groups is
efficiently boosted by HIV-VLP induction but does not switch
into a Th1 pattern.
MATERIALS AND METHODS
Cell culture medium. PBMC culture medium consisted of RPMI 1640 medium
(Life Technologies, Carlsbad, CA) supplemented with 2 mM L-glutamine
(Sigma), 1% nonessential amino acids (Life Technologies), 1% sodium pyruvate
(Life Technologies), 50 ␮M 2-mercaptoethanol (Sigma), 50 ␮g of gentamicin
(Life Technologies) per ml, and 10% fetal calf serum (Life Technologies).
PBMC preparations and cell treatment. All human specimens were obtained
and processed at the Institute of Human Virology in Baltimore, MD, with
informed consent, as approved by the University of Maryland—Baltimore Insti-
tutional Review Board. Fresh human PBMCs were isolated by Ficoll-Hypaque
density gradient centrifugation and plated in six-well plates at a concentration of
approximately 1 ⫻10
7
/well in a maximum volume of 3 ml/well. PBMCs were
pulsed with 6 ␮g/ml of HIV-VLPs. As a negative control, PBMCs were treated
with phosphate-buffered saline (PBS). The residual endotoxin activity possibly
present in the HIV-VLP preparation was inhibited by preincubation with poly-
myxin B sulfate (Sigma) at a concentration of 10 ␮g/ml. The absence of inter-
ference due to polymyxin B sulfate in the activation results was verified in
parallel with PBMCs treated only with polymyxin. After 16 h, the cells were
harvested, washed, and stained for phenotypic analysis by flow cytometry. The
cellular supernatants were collected for quantification of cytokine production by
enzyme-linked immunosorbent assay (ELISA) (University of Maryland Cytokine
Core Lab, Baltimore, MD). During the development/optimization of the exper-
imental assay, the absence of PBMC activation following treatment with either
PBS, supernatant of SF9 cell culture transfected with a baculovirus expression
vector (mock baculovirus supernatants), or heat-denatured VLP suspension in
PBS (100°C for 10 min) has been repeatedly observed. Thus, the complete set of
experiments was subsequently performed using only the PBS treatment as a
negative control.
Flow cytometry. Cells were incubated for 30 min at 4°C with the indicated
murine monoclonal antibodies (BD Pharmingen, San Diego, CA), washed, and
then fixed with 2% paraformaldehyde for analysis with a FACScalibur flow
cytometer (BD Pharmingen). Data analysis was carried out using the FlowJo
software program (Tree Star Inc., San Carlos, CA). All analyses were performed
on freshly isolated PBMCs, and levels of activation markers were evaluated for
cell populations gated for CD14 positivity (high or dim). The fraction of PBMCs
that responded by upregulation of activation markers on the cell surface was
calculated by overlaying the histograms of treated and untreated PBMCs and
carrying out Overton subtraction of the curves.
Statistical analyses. Intergroup comparisons were performed using the Mann-
Whitney U test (for univariate nonparametric group analysis). All Pvalues were
two-tailed and were considered significant if less than 0.05.
RESULTS
Clinical parameters of subjects included in analysis. Twenty-
three subjects were enrolled in the study. Nineteen were HIV-1
seropositive, of whom 11 showed a low HIV-1 viremia (⬍2.6
log RNA copies/ml) and 8 a high viremia (⬎4.69 log RNA
copies/ml); the latter enrolled at their first visit and were naive
for ART treatment. In particular, the log of viral RNA copies
per milliliter ranged from 1.34 to 2.54 (mean, 107 copies/ml)
for the low-viremia group and from 4.74 to 5.92 (mean, 295,120
copies/ml), for the high-viremia group (P⬍0.0001). The
CD4
⫹
counts ranged from 29 to 532 cells/mm
3
(mean, 240
cells/mm
3
) for the low-viremia group and from 2 to 296 cells/
mm
3
(mean, 117 cells/mm
3
) for the high-viremia group (P⫽
0.0298) (Fig. 1). Four healthy seronegative subjects were en-
rolled as controls.
Baculovirus HIV-VLPs induce maturation phenotype in
CD14
ⴙ
PBMCs. Human PBMCs were isolated by Ficoll-
Hypaque density gradient centrifugation, plated in six-well
plates at a concentration of 1 ⫻10
7
/well, and incubated with 6
␮g/ml of HIV-VLPs. In parallel, PBMCs were incubated with
PBS as a negative control. After 16 h, the expression of the sur-
face maturation markers CD80, CD83, CD86, and HLA-DR
was examined in the CD14
⫹
monocyte population, whose dis-
tribution was comparable between seronegative and seroposi-
tive subjects, in both the CD14
hi
and CD14
dim
cellular subsets.
The basal expression of the four markers, in terms of mean
fluorescence index (MFI), is largely comparable between se-
ronegative and seropositive subjects, regardless of the HIV-1
viremia levels, in both CD14
hi
and CD14
dim
monocyte popu-
lations (Fig. 2A to D). The only exception is represented by
basal CD83 expression, which is significantly lower in CD14
dim
monocyte populations of the low-viremia seropositive group
(Fig. 2A).
VOL. 83, 2009 Th2 POLARIZATION IN PBMCs BY HIV-VLP 305

Similarly, the HIV-VLP-induced expression of the four
markers is comparable between the seronegative and seropos-
itive subjects, regardless of HIV-1 viremia levels, in both
CD14
hi
and CD14
dim
monocyte populations (Fig. 3A to D).
This observation is further confirmed by the MFI activation for
each maturation marker (VLP versus basal), suggesting that
overall the seropositivity status, with either low or high viremia,
does not significantly affect the responsiveness to an immuno-
FIG. 1. Clinical parameters of subjects included in the analysis. The individual values of viremia and CD4
⫹
cell counts observed for the enrolled subjects
are reported. Means and standard errors for each group (low-viremia and high-viremia) are shown. ⫺ve, negative; ⫹ve, positive; n.a., not applicable.
FIG. 2. Basal expression of activation markers and costimulatory molecules in CD14
⫹
cells (A to D). The basal expression of CD80, CD83,
CD86, and HLA-DR on PBMCs was analyzed on fixed cells by using a FACScalibur flow cytometer, and data analysis was carried out using the
FlowJo software program. PBMCs were gated for CD14 positivity. The MFI for each sample in each group is represented in both CD14
dim
and
CD14
hi
gating. Means and standard errors for each group are shown.
306 BUONAGURO ET AL. J. VIROL.

gen stimulus (i.e., HIV-VLPs) of circulating monocyte CD14
⫹
cell populations (Fig. 4A to D).
In the panels for both basal and HIV-VLP-induced expres-
sion of the four markers, “hyperresponsive” subjects are
present in low- as well as high-viremia groups (Fig. 2 to 4),
although none of them is consistently an “outlier” for all four
markers evaluated in this study.
Differential maturation phenotype HIV-1-seropositive sub-
jects. HIV-VLP-induced maturation phenotypes for individual
HIV-1-seropositive subjects were evaluated in both low- and
high-viremia groups. The results indicate that a clear matura-
tion phenotype is observed for the majority of subjects, regard-
less of the levels of HIV-1 viremia; nevertheless, it is also
evident that for a couple of subjects in both groups, a lack of
maturation induced by HIV-VLPs in CD14
⫹
circulating cells
was observed when CD14
hi
as well as CD14
dim
monocyte pop-
ulations were considered (Fig. 5 and 6).
In particular, in the low-viremia group, subjects 5 and 14
showed a complete lack of activation for all four markers
evaluated in both CD14
hi
and CD14
dim
monocyte populations,
while subject 17 showed such an “anergic” phenotype only in
the CD14
hi
monocyte population (Fig. 5A and 6A). Interest-
ingly, the very low viremia (1.44 to 1.61 log RNA copies/ml)
and the relatively normal CD4
⫹
cell count (173 to 532 cells/␮l)
observed for these subjects would not predict any impairment
in response to an antigen.
Similarly, in the high-viremia group, subjects 12 and 15
showed an incomplete activation of the four markers evaluated
in both CD14
hi
and CD14
dim
monocyte populations (Fig. 5B
and 6B). In particular, subject 15 showed activation only of the
CD86 costimulatory molecule, which by itself has been shown
to not be sufficient to drive T-cell immunity (26, 53, 55). Sub-
ject 12, instead, showed activation of costimulatory molecules
(in particular, HLA-DR and CD80) only in a subpopulation of
CD14
dim
monocytes (Fig. 5B).
These two subjects showed the highest viremia in this group
(5.76 and 5.92 log RNA copies/ml), associated with a low
CD4
⫹
cell count (62 to 164 cells/␮l), suggesting in the high-
viremia group a possible correlation between higher viremia
and an impaired response to an antigen, although further stud-
ies of a larger cohort of subjects will be needed to confirm this
preliminary observation.
Cytokine production in HIV-VLP-loaded PBMCs. In order
to evaluate the impact of chronic HIV infection on the pro-
duction of cytokines important for T-helper-cell activation, the
levels of IL-12 p70, gamma interferon (IFN-␥), tumor necrosis
factor alpha (TNF-␣), IL-6, IL-4, and IL-10 were assessed in
the supernatant of PBMCs loaded with HIV-VLPs.
FIG. 3. Expression of activation markers and costimulatory molecules induced by HIV-VLPs in CD14
⫹
cells (A to D). PBMCs were incubated
in the presence of HIV-VLPs for 16 h. The expression of CD80, CD83, CD86, and HLA-DR was analyzed on fixed cells by using a FACScalibur
flow cytometer, and data analysis was carried out using the FlowJo software program. PBMCs were gated for CD14 positivity. The MFI for each
sample in each group has been represented in both CD14
dim
and CD14
hi
gating. Means and standard errors for each group are shown.
VOL. 83, 2009 Th2 POLARIZATION IN PBMCs BY HIV-VLP 307

The average basal level of all evaluated cytokines was low
(⬍20 pg/ml), with no significant difference between HIV-1-
seronegative and -seropositive individuals; moreover, in the
latter group, the level of viremia did not make a significant
difference. The only exception was represented by IL-6, whose
average basal levels in both the low- and high-viremia seropos-
itive groups (51.06 and 64.66 pg/ml, respectively) were signif-
icantly higher than that in the seronegative group (12.37 pg/ml)
(Fig. 7).
Treatment of PBMCs with HIV-VLPs did not induce any
increase in the production of IL-12 p70 or IL-4, regardless of
the HIV-1 serological status (Fig. 8A). On the contrary, HIV-
VLPs induced a significantly increased production of the other
Th2 cytokines in HIV-seronegative and -seropositive samples,
with an average 1- to 2-logfold increase for IL-10, IL-6, and
TNF-␣. However, the increased production of IL-10 and
TNF-␣in the high-viremia seropositive group was significantly
lower than those in the other groups (P⬍0.05) (Fig. 8B).
Moreover, the production of the Th1 IFN-␥was significantly
increased of 2.5-fold by HIV-VLP treatment only in the
healthy seronegative group, while in the seropositive groups
the observed increased production was not statistically signif-
icant (P⬎0.05) (Fig. 8B).
DISCUSSION
We have previously reported that baculovirus-expressed
HIV-VLP
A
s, which display gp120 derived from a Ugandan
HIV-1 isolate of the subtype A, are strongly immunogenic in
BALB/c mice, inducing HIV-1-specific CD4
⫹
and CD8
⫹
T-cell
responses as well as cross-clade neutralizing antibodies at sys-
temic and mucosal sites (9, 11, 13). We have also shown that
these HIV-VLPs are able to induce maturation of ex vivo-
generated immature human MDDCs, resulting in the expres-
sion of surface maturation markers and the increased produc-
tion of Th1 and Th2 cytokines, and this observation can be
confirmed on unselected PBMCs (1, 10).
Here we show that the basal and HIV-VLP-induced ex-
pression of CD80, CD83, CD86, and HLA-DR activation
markers and costimulatory molecules, in terms of mean
fluorescence, is largely comparable between seronegative
and seropositive groups, regardless of HIV-1 viremia levels,
in both CD14
hi
and CD14
dim
monocyte populations. The
overall expression pattern suggests maturation/activation in-
duced by VLPs, although for some specific markers and in
some patients, the trend does not reach statistical signifi-
cance. This observation suggests that the seropositivity sta-
tus, with either low or high viremia, does not significantly
FIG. 4. n-fold induction of activation markers and costimulatory molecules by HIV-VLPs in CD14
⫹
cells (A to D). The n-fold increase in the
MFI observed for each sample in each group after induction with HIV-VLPs, over the basal level (VLP versus PBS), is represented in both
CD14
dim
and CD14
hi
gating. Means and standard errors for each group are shown.
308 BUONAGURO ET AL. J. VIROL.

impair the immune activation status and the responsiveness
of circulating monocyte CD14
⫹
cell populations to an im-
munogenic stimulus. Results obtained in parallel, with lipo-
polysaccharide used as a positive control, confirm the stim-
ulation effects on PBMCs from the three groups analyzed in
the present study (M. Monaco, personal communication).
Nevertheless, it is also evident that in both the low-vire-
mia and high-viremia seropositive groups, some individuals
show a complete lack of maturation induced by HIV-VLPs
in CD14
⫹
circulating cells, with both the CD14
hi
and
CD14
dim
monocyte populations being considered. Such an
“anergic” phenotype, at least in the low-viremia HIV-1-
seropositive group, does not consistently correlate to an
advanced status of HIV-1 infection (i.e., low CD4
⫹
cell
count and high viremia), indicating the need for individual
evaluations to identify possible impairments in response to
an immunogen.
The present results confirm and extend data from others
showing a normal expression of surface molecules involved in
antigen-specific T-cell activation on immature and mature DCs
from HIV-1-infected and hepatitis C virus (HCV)-HIV-coin-
fected individuals (15, 25, 31). Furthermore, monocyte-derived
DCs from either HCV-infected or HCV-HIV-coinfected per-
sons have been previously shown to stimulate a mixed leuko-
FIG. 5. Individual pattern of activation in CD14
dim
cells. The expression of CD80, CD83, CD86, and HLA-DR in PBMCs was analyzed on fixed
cells by using a FACScalibur flow cytometer, and data analysis was carried out using the FlowJo software program. The PBMCs were gated for
CD14
dim
positivity. Results of a representative experiment are shown; the shaded curve represents the baseline data for untreated cells.
(A) Expression of markers in low-viremia group. (B) Expression of markers in high-viremia group. In each panel, the arrow indicates the samples
with no induction of expression.
FIG. 6. Individual pattern of activation in CD14
hi
cells. The expression of CD80, CD83, CD86, and HLA-DR in PBMCs was analyzed on fixed
cells by using a FACScalibur flow cytometer, and data analysis was carried out using the FlowJo software program. PBMCs were gated for CD14
dim
positivity. The results of a representative experiment are shown; the shaded curve represents the baseline data for untreated cells. (A) Expression
of markers in low-viremia group. (B) Expression of markers in high-viremia group. In each panel, the arrow indicates the samples with no induction
of expression.
VOL. 83, 2009 Th2 POLARIZATION IN PBMCs BY HIV-VLP 309

cyte reaction in purified, allogeneic CD4
⫹
T cells comparable
to that with DCs derived from healthy donors (36, 44, 54).
The average basal level of the IL-12 p70, IFN-␥, TNF-␣,
IL-6, IL-4, and IL-10 cytokines, relevant for T-helper cell ac-
tivation and polarization, is low (⬍20 pg/ml) in HIV-1-sero-
positive individuals, as in healthy seronegative subjects. The
only exception is represented by IL-6, whose average basal
level in both low- and high-viremia seropositive groups is four
to fivefold higher than that in the seronegative group. Given
that IL-6 promotes Th2 differentiation (20) and inhibits IFN-␥
production and Th1 differentiation (19), the higher basal levels
of IL-6 in HIV-seropositive individuals suggest a Th2 polar-
ization induced by an established HIV-1 infection, regard-
less of the viremia levels, as previously extensively reported
(5, 16, 24).
Interestingly, in both HIV-seropositive groups, mainly Th2-
polarizing cytokines (IL-6, IL-10, and TNF-␣) were induced by
HIV-VLPs, while the increased production of Th1 IFN-␥was
not statistically significant (P⬎0.05), suggesting the persis-
tence of a prevalent Th2 status. However, the significantly
lesser increase in production of IL-10 and TNF-␣in the high-
viremia seropositive group suggests a progressively functional
impairment associated with an advanced status of HIV-1 in-
fection. No significant difference in the secretion of the Th2
cytokine TNF-␣or IL-10 was observed between PBMCs from
healthy and low-viremia HIV-seropositive individuals, as pre-
viously reported by others in different experimental systems
(14, 39).
The production of IL-4 and IL-12 p70 was not increased by
HIV-VLPs in the seronegative or seropositive group. In par-
ticular, the production of IL-12 p70 by monocyte/macrophage
cells and B lymphocytes is known to be inhibited by IL-10 and
TNF-␣(2, 17, 37), with a sequential detrimental effect on the
IL-12-mediated induction of IFN-␥production by NK and T
cells (34, 35, 41). Therefore, the high levels of IL-10 and
TNF-␣induced by HIV-VLPs could explain the lack of in-
creased production of IL-12 p70 in all tested groups and the
limited production of IFN-␥only in the seronegative group.
The impairment of basal and antigen-induced production of
Th1-polarizing cytokines for HIV-seropositive individuals is in
concordance with previous observations of PBMCs from HIV-
infected subjects exposed ex vivo to a panel of stimuli (i.e.,
Staphylococcus aureus, bacterial cell wall components) (25, 39).
The correlation of the lymphokine response with the extent of
stimulation of CD83, CD86, CD80, and HLA-DR markers for
each subject of each group is currently under evaluation.
These results represent a proof-of-concept of the approach
and have been generated using Env-presenting HIV-VLPs;
additional VLPs and particulate structures (i.e., virosomes)
(with or without presenting Env) are currently under evalua-
tion. They show that in order to obtain a therapeutic effect in
HIV-infected individuals, it would be mandatory to reduce the
FIG. 7. Basal expression of Th1 and Th2 cytokines in PBMCs. The basal expression of IL-12 p70, IFN-␥, TNF-␣, IL-6, IL-4, and IL-10 by
PBMCs was measured by ELISA in the culture supernatant. The value for each sample in each group is shown. Means and standard errors for
each group are shown.
310 BUONAGURO ET AL. J. VIROL.

FIG. 8. Expression of Th1 and Th2 cytokines induced by HIV-VLPs in PBMCs (A and B). PBMCs were incubated in the presence of
HIV-VLPs for 16 h, and expression of IL-12 p70, IFN-␥, TNF-␣, IL-6, IL-4, and IL-10 by PBMCs was measured in the culture supernatant
by ELISA. For each group, the PBS (basal)- and VLP-induced (VLP) levels are reported. The value for each sample in each group is shown.
The mean and SEM of the values in each group are shown.
VOL. 83, 2009 Th2 POLARIZATION IN PBMCs BY HIV-VLP 311

viremia and to use adjuvanting molecules able to shift the
immune system toward a Th1-cell-mediated immune response
to override the established Th2 status. Otherwise, the thera-
peutic vaccination would fail either for lack of effective activa-
tion of Th1/Th2 cells or because of detrimental further boost-
ing of a Th2 response, useless for a therapeutic effect and
inhibiting the possible ongoing Th1 response.
The overall results presented here show the possibility of
screening donor susceptibility to an antigen treatment using
PBMCs without the need of purification and ex vivo selection
of DCs, simplifying the identification of “responsive” vaccinees
and the understanding of eventual failures in individuals en-
rolled in clinical trials. When necessary, additional and more-
detailed studies of fractionated cell types would allow identi-
fication and a better characterization of the individual cells
involved in the in vivo response. This still-limited set of mark-
ers, which could be further broadened, has proven to be infor-
mative for identifying subjects who, in the genomic study de-
scribed in a parallel work, are confirmed to be totally or
partially anergic (Monaco, personal communication).
In conclusion, our results indicate that monocyte-derived
DCs from HIV-1-seropositive patients retain a strong APC
function and can be functional in active autologous immuno-
therapy strategies. However, specific Th1-driving adjuvant
strategies might be necessary to obtain the efficient therapeutic
effect sought.
ACKNOWLEDGMENTS
We are indebted to Robin Flinko for invaluable technical support.
This study was supported by grants from the Ministero Italiano
Universita` e Ricerca (MIUR, 2004), the Ministero Italiano della
Sanita` (Ricerca Corrente and Progetto Finalizzato AIDS 2006), the
Institute of Human Virology, and the NIH (NIH grants to G.K.L.).
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