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Biomarkers of immune dysfunction following
combination antiretroviral therapy for
HIV infection
Combination antiretroviral therapy (cART) has significantly reduced morbidity and mortality of HIV‑infected
patients, yet their life expectancy remains reduced compared with the general population. Most
HIV‑infected patients receiving cART have some persistent immune dysfunction characterized by chronic
immune activation and premature aging of the immune system. Here we review biomarkers of T‑cell
activation (CD69, -25 and -38, HLA-DR, and soluble CD26 and -30); generalized immune activation
(C‑reactive protein, IL‑6 and D‑dimer); microbial translocation (lipopolysaccharide, 16S rDNA,
lipopolysaccharide-binding protein and soluble CD14); and immune dysfunction of specific cellular subsets
(T cells, natural killer cells and monocytes) in HIV-infected patients on cART and their relationship to
adverse clinical outcomes including impaired CD4 T-cell recovery, as well as non-AIDS clinical events, such
as cardiovascular disease.
KEYWORDS: antiretroviral therapy n HIV n immune activation n immune dysfunction
n immune senescence n microbial translocation n non-AIDS illness
A large number of biomarkers have been evaluated in HIV-infected patients and have largely
been used to predict clinical outcome, including
AIDS and death. Given the widespread availability of combination antiretroviral therapy (cART)
and the ability of cART to reduce morbidity
and mortality, this article primarily focuses on
biomarkers of interest in HIV-infected patients
receiving cART, with an emphasis on the effect
of microbial translocation and immune activation. Following cART, many patients continue
to demonstrate ongoing immune abnormalities
and patients are at an increased risk of non-AIDS
events including cardiovascular disease (CVD)
and malignancy. Some of these biomarkers are in
early clinical development while others have been
evaluated in larger clinical trials and cohorts to
determine whether they can predict adverse clinical outcomes in patients on cART. Most of these
biomarkers are not yet used in routine clinical
practice but are currently being evaluated to
understand the pathogenesis of immune dysfunction in patients on cART, identify patients
at highest risk of immune dysfunction and nonAIDS events, such as CVD and malignancy, and
develop novel immunotherapeutic approaches
that could be used in addition to cART.
HIV pathogenesis & response
to cART
The physiological hallmark of HIV infection
is the destruction of CD4 + T cells and persistent immune activation. HIV predominantly
infects activated CD4 + T cells, resulting in
productive viral infection [1] , and a rapid and
profound depletion of GI tract-associated
CD4 + T cells, leading to loss of integrity of
the intestinal mucosa [2,3] . The depletion of
GI tract CD4 + T cells has been associated with
increased translocation of microbial products
from the intestinal lumen into the blood stream,
which can trigger the innate immune system
to produce proinflammatory cytokines and
chronic immune activation [3] . However, the
pathogenesis of immune activation is complex
and multifactorial. In addition to the translocation of microbial products, immune activation
is believed to be driven by direct infection of
CD4 + T cells and release of cytokines, a combination of innate and adaptive immune responses
against viral antigens and/or secondary infections, and an imbalance in the immune response
caused by the loss of T regulatory and central
memory T cells (reviewed in [4]).
Combination antiretroviral therapy has led to
a dramatic reduction in mortality and morbidity
in HIV-infected patients [5,6] . Despite long-term
suppression of HIV RNA and recovery of the
total number of CD4 + T cells in most patients
following cART, full life expectancy of patients
is restored in some [7] , but not all, patients [6,8] .
The chance of an HIV-infected patient reaching the age of 70 years is still approximately
50% that of HIV-uninfected controls [8] . HIVinfected patients experience higher rates of
CVD, non-AIDS malignancies, dementia, liver
10.2217/BMM.11.15 © 2011 Future Medicine Ltd
Biomarkers Med. (2011) 5(2), 171–186
Gregor F Lichtfuss1,2,
Jennifer Hoy2,3,
Reena Rajasuriar1,2,4,
Marit Kramski5,
Suzanne M Crowe1,2,3
& Sharon R Lewin†1,2,3
Centre for Virology, Burnet Institute,
Melbourne, Australia
2
Department of Medicine, Monash
University, Melbourne, Australia
3
Infectious Diseases Unit, The Alfred
Hospital, Melbourne, Australia
4
Faculty of Medicine, University of
Malaya, Kuala Lumpur, Malaysia
5
Department of Microbiology
& Immunology, University of
Melbourne, Melbourne, Australia
†
Author for correspondence:
Tel.: +61 390 768 491
[email protected]
1
ISSN 1752-0363
171
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Lichtfuss, Hoy, Rajasuriar, Kramski, Crowe & Lewin
disease, renal diseases and bone disorders [9,10] .
Factors that drive ongoing morbidity include a
combination of immune activation, aging of the
immune system and cART toxicity (reviewed
in [11]).
Biomarkers in HIV-infected patients
& the effect of cART
„„
Immune activation
A detailed overview of markers of persistent
immune activation in HIV-infected patients
has recently been published [12] . Here we will
focus only on measures of immune activation in
patients receiving cART (summarized in Table 1)
with a focus on larger prospective studies that
have aimed to identify associations between the
relevant biomarker and clinical outcomes.
T‑cell activation: cellular markers CD69,
-25 & -38, & HLA-DR
T‑cell activation can be monitored using either
cell-associated or soluble markers. T‑cell activation is identified by increased expression of
CD69, -25 and -38, and HLA-DR. CD69,
a transmembrane C‑type lectin, is an early
marker of activation [13] and is followed by
increased expression of CD25, the a‑chain of
the IL‑2 receptor [14] . The MHC class II antigen, HLA-DR, is expressed in later phases of
T‑cell activation, while CD38 (an extracellular
ADP-ribose cyclase) is expressed constitutively
in activated T cells [15] .
CD69 expression is a useful tool to monitor
T‑cell activation in vitro [16] ; however, expression of CD69 from freshly isolated blood is
low and is similar in HIV-infected and HIVuninfected patients [16,17] , potentially because
of the transient expression of these markers or
sequestration of CD69 + T cells in lymph nodes
and other organs. CD25, although a marker of
T‑cell activation and used as such in vitro, is
also expressed on regulatory T cells [18] , often
in combination with other intracellular markers
such as FoxP3 [19] . Most studies characterizing
T‑cell activation during HIV infection in vivo
have therefore focused on expression of CD38
and HLA-DR [20–22] .
Increased expression of CD38 and HLA-DR
on CD4 + and CD8 + T cells in untreated HIV
infection has been associated with shorter
survival and faster disease progression [23–25] .
However, expression of CD38 on CD8 + T cells
before the initiation of cART was not predictive for clinical outcomes in patients receiving
cART [26] . Following cART, expression of CD38
and HLA-DR on both CD4 + and CD8 + T cells
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Biomarkers Med. (2011) 5(2)
declines significantly but remains higher than
in uninfected controls [27] . Persistent elevation
of CD38 and HLA-DR on both CD4 + and
CD8 + T cells has been associated with impaired
magnitude and speed of CD4 + T‑cell recovery [27–29] , and higher levels of expression of
CD38 on CD8 + T cells was observed in patients
on cART at the time of an opportunistic infection or death [26] . T‑cell activation markers were
significantly higher in HIV-infected patients
from Africa compared with the USA and were
associated with poorer CD4 + T‑cell recovery
and increased mortality following cART in this
Ugandan-based prospective study [30] .
T‑cell activation: soluble markers
Plasma markers for T‑cell activation are of potential interest, as these can be more easily measured
than cell-associated markers in clinical practice.
Activated T cells express a number of proteins on
their surface that are shed, including CD26, -27
and -30, and the ligand of the CD40 receptor,
leading to increased levels of their soluble forms
in plasma.
Th1 cells predominantly express and release
CD26, while Th2 cells express and release
CD30. Therefore, these plasma markers have
been investigated in conditions that are defined
by either a Th1 [31] or a Th2 environment [32,33] .
While soluble (s)CD30 is directly quantified in
plasma using a standard ELISA, sCD26 is usually indirectly detected through its enzymatic
activity [34] .
A decrease of enzymatic activity of sCD26
had been inversely correlated with HIV RNA
in untreated HIV-infected patients [35] . In
patients receiving cART, there was no significant
elevation in sCD26 compared with uninfected
individuals, regardless of whether patients had
detectable HIV RNA [34] . Despite the potential use of sCD26 as a diagnostic or prognostic marker in other diseases [36] , its value as a
marker in the context of HIV infection has
not been established and, therefore, requires
further investigation.
Plasma levels of sCD30 are elevated during
the early phase of HIV infection and decrease
following seroconversion, correlating with
detectable HIV p24 antigen [37] , and were
associated with faster progression to AIDS in
untreated HIV-infected patients [38,39] . Data
regarding changes in sCD30 in untreated HIVinfected patients are inconsistent, with some
studies reporting higher levels [40,41] or levels
similar to uninfected subjects [34] . However, a
recent large retrospective cohort study (n = 290)
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Biomarkers of immune dysfunction following combination antiretroviral therapy
demonstrated a significant decrease of sCD30
following initiation of cART, even in the absence
of virological control. sCD30 also correlated
with hypergammaglobulinemia, an indicator of
B-cell activation [42] . In a separate cohort study
of patients receiving cART (n = 276), higher
baseline levels of sCD30 prior to cART initiation were associated with higher HIV RNA [43] .
In patients who achieved virological suppression
(<500 copies/ml), a faster decrease of sCD30 in
the first 6 months predicted a higher risk of subsequent viral rebound [43] . To date, no studies
have evaluated the relationship between sCD30
and clinical outcome on cART.
Receptors of the TNF receptor (TNFR)
superfamily include CD27 expressed on T cells,
CD40 expressed on antigen presenting cells and
TNFR‑1, which is widely expressed across cell
types. The TNFR superfamily are involved in
pathways regulating a wide array of cellular functions, such as cell proliferation, differentiation
and survival, and are often shed following receptor engagement. The CD40 ligand (CD40L) is
expressed and shed by activated T cells. Plasma
levels of sCD27 [44,45] and sCD40L are elevated
in untreated HIV infection [46] .
Combination antiretroviral therapy leads
to a significant reduction of plasma levels of
sCD27 [42] , while increased levels of sCD40L
do not appear to be affected by cART [47] .
In a recent case–control study of treatmentnaive patients initiating cART, pretreatment
levels of sTNFR‑1, sCD27 and sCD40L were
each significantly associated with the risk of an
AIDS-defining malignancy or death [26] . These
clinical events presented a median of 51 weeks
after cART initiation, by which time the majority of subjects had over 200 circulating CD4
cells/mm3 and had suppressed plasma viremia to
less than 50 copies/ml. Therefore, these markers
may potentially identify patients at the highest
risk of an AIDS-defining malignancy or death
on cART [26] .
Generalized immune activation &
thrombotic risk: C-reactive protein, IL‑6
& D‑dimer
C-reactive protein (CRP), IL‑6 and D‑dimer
have been extensively studied as biomarkers
of persistent immune activation and increased
thrombotic risk in patients on cART [12] . The
interest in these biomarkers started following
the Strategic Management of Antiretroviral
Therapy (SMART) study. The SMART study
was a random­ized controlled trial comparing the
effect of intermittent CD4-guided cART versus
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Special Report
continuous cART on morbidity and mortality [48] . SMART was one of the first studies to
identify a relationship between viral replication,
immune activation and non-AIDS events, and
several substudies of SMART have identified
multiple biomarkers that are associated with
non-AIDS events [49–53] and mortality [54] .
C-reactive protein, generally measured
using a high-sensitivity assay and, therefore,
referred to as highly sensitive (hs)CRP, is an
acute-phase protein produced by hepatocytes in
response to IL‑6 and is involved in complement
formation [55] . Elevated CRP has been associated with CVD in over 30 prospective cohort
and case–control studies in previously healthy
middle-aged and elderly populations [56,57] . In
the setting of HIV infection, levels of hsCRP
are higher than age- and sex-matched HIVnegative controls [52] , and increased levels of
CRP have been demonstrated to be associated
with a greater risk of development of opportunistic infection [50,58] , and cardiovascular or allcause mortality [49,59] ; although the association
with cardio­vascular risk was not reported in all
studies [49,59,60] . Following initiation of cART,
most studies have reported little or no change
in hsCRP, although one study demonstrated
a significant reduction in hsCRP [42,51,61–63] .
However, increased levels of hsCRP in plasma
of patients receiving cART was associated with
increased carotid intima media thickness, a
predictor for CVD [64] .
IL‑6 is a proinflammatory cytokine produced
mainly by monocytes, endothelial cells and
lympho­c ytes, [65] . IL‑6 levels increase with age
in the general population and have been associated with increased CVD and mortality in the
elderly [57,66,67] . Compared with the general population, IL‑6 levels are significantly increased in
HIV infection [52] . In untreated HIV-infected
patients, IL‑6 levels are elevated and associated
with markers of vascular dysfunction (reduced
small artery elasticity) and some plasma markers of endothelial function, including E-selectin
and soluble ICAM‑1 [52,68] . As with the observations for hsCRP, cART did not lead to a significant reduction in IL‑6 [51] , not even after
3 years of continuous cART [42] . However, when
cART was interrupted, IL‑6 levels significantly
increased, and the magnitude of the increase correlated with levels of HIV RNA [49] . Increased
baseline levels of IL‑6, before the initiation of
cART, was the strongest predictor of all-cause
mortality in the SMART study [49] and was also
associated with the development of opportunistic
disease [26,50] .
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Lichtfuss, Hoy, Rajasuriar, Kramski, Crowe & Lewin
Table 1. Biomarkers commonly used to assess HIV-infected patients on combination antiretroviral therapy and
their relationship with clinical outcomes.
Biomarker
Marker
Assay type
Specimen
Function
HIV infection
Naive
On cART
Reduced but slow/no
normalization
Unclear
T‑cell activation
CD38/HLA-DR
–
Flow cytometry
WB/PBMC
sCD26
(Th1)
Plasma
sCD27
sCD30
sCD40L
sTNFR‑1
–
(Th2)
–
–
Enzymatic activity
assay
ELISA
ELISA
ELISA
ELISA
Elevated on CD4 +
and CD8 + T cells
Reduced
Plasma
Plasma
Plasma
Plasma
Elevated
Elevated
Elevated
Elevated
Reduced
Reduced/elevated in IRD
No change
–
Inflammation/immune activation
CRP
IL‑6
–
–
ELISA
ELISA
Plasma
Plasma
Elevated
Elevated
Reduced in some studies
No change
D‑dimer
–
ELISA
Plasma
Elevated
–
LPS
–
Enzymatic activity
assay
Plasma
Elevated
Reduced but slow/no
normalization
16S rDNA
–
PCR
Plasma
Elevated
Reduced
Microbial translocation
Immune response to bacterial products
LBP
–
ELISA
Plasma
Elevated
–
sCD14
–
ELISA
Plasma
Elevated
EndoCAb
–
ELISA
Plasma
Elevated
Reduced but slow/no
normalization
Reduced
CD28/CD57
Senescence
Flow cytometry
WB/PBMC
Elevated
No normalization
Antigen-specific
dysfunction
Against CMV
Abnormal immune
restoration
–
–
–
–
–
Functional assay†
WB/PBMC
Elevated
Elevated
Against Mtb
–
Functional assay†
WB/PBMC
Reduced
Against HBV
–
Functional assay†
WB/PBMC
Reduced
Variable increase, can be
excessive
No normalization
Immune activation
Flow cytometry
WB/PBMC
Elevated
No normalization
T‑cell dysfunction
NK cell dysfunction
HLA-DR on NK
All associations are positive correlations unless otherwise specified.
†
Incubation with specific antigen or peptides and cytokine production measured by enzyme-linked immunospot (ELISpot) or intracellular cytokine staining.
‡
Functional assay requires ex vivo stimulation and intracellular staining.
§
CD107a expression elevated if measured by surface expressioin ex vivo but reduced if measured in response to experimental stimulus.
¶
Cell bound TF measured by flow cytometry. sTF requires an Enzymatic Activity Assay.
ADCC: Antibody-dependent cell cytotoxicity; BMD: Bone mineral density; cART: Combination antiretroviral therapy; CMV: Cytomegalovirus; CRP: C-reactive protein;
CVD: Cardiovascular disease; EndoCAb: Endotoxin core antibody; HBV: Hepatitis B virus; HCV: Hepatitis C virus; IRD: Immune restoration disease;
LBP: Lipopolysaccharide-binding protein; LPS: Lipopolysaccharide; Mtb: Mycobacterium tuberculosis; NHL: Non-Hodgkin’s lymphoma; NK: Natural killer;
PBMC: Peripheral blood mononuclear cell; s: Soluble; TF: Tissue factor; TNFR: TNF receptor; WB: Whole blood.
174
Biomarkers Med. (2011) 5(2)
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Biomarkers of immune dysfunction following combination antiretroviral therapy
Association with other biomarkers
Naive
Association with clinical outcome
Ref.
Naive (HIV )
On cART (HIV )
HIV un‑infected
LPS, 16S rDNA, sCD14, inverse
–
correlation with CD28/CD57, TF
Inverse correlation with HIV RNA –
Mortality, disease
progression
–
–
–
[23–29]
–
–
[34–36]
sTNFR‑1, IL‑6, CD4 count
HIV RNA
sTNFR‑1
sCD27, IL‑6, HIV RNA inverse
correlation with CD4 count
–
–
–
–
–
NHL
–
–
–
–
–
–
–
NHL
–
–
HIV RNA, CD4 count
–
–
–
TF
HIV RNA,
TF
–
Vascular dysfunction,
endothelial
dysfunction
–
CVD, mortality
CVD, BMD
Mortality,
Age, mortality
opportunistic disease and NHL, reduced
BMD
Mortality,
CVD, mortality
opportunistic disease
16S rDNA, LBP, sCD14, CD8 +
T‑cell activation
16S rDNA,
sCD14,
CD8 + T-cell
activation
LPS, CD8 +
T-cell
activation
Opportunistic
infection, dementia,
liver disease
progression (HCV)
–
Inverse correlation
with CD4 + T‑cell
recovery
–
[3,77,80–89,91,101]
Inverse correlation
with CD4 + T‑cell
recovery
–
[77]
LPS, sCD14
–
–
–
[3,91]
LPS, CD8 + T‑cell activation,
NK activation
Inverse correlation with LPS
LPS
Faster CD4 + T‑cell
recovery
Mortality
Faster disease
progression, dementia
–
–
–
[3,48,54,78,83,89–91,94,95]
–
[3,54]
Inverse correlation with HLA-DR –
on CD4 + T‑cells
–
Increase of CD28 with AIDS
–
Lower arterial
distensibility
–
Aging
–
–
–
CVD
–
–
–
Carotid intima
thickness, CVD
Severe Mtb disease
–
[118,119]
–
–
–
–
–
[117]
sCD14
–
–
–
–
[Lichtfuss GF, Lewin SR,
HIV RNA, CD8 + T‑cell activation
On cART
Special Report
–
+
+
[26,42,44–46]
[34,37–43]
[26,46,47]
[26]
[42,49,50,52,56–64]
[26,42,49–52,57, 6–68]
[49–52,69,71–73]
[105,106, 111–114,160]
–
[121–125]
Jaworwski A, Crowe SM,
Unpublished Data]
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Lichtfuss, Hoy, Rajasuriar, Kramski, Crowe & Lewin
Table 1. Biomarkers commonly used to assess HIV-infected patients on combination antiretroviral therapy and
their relationship with clinical outcomes (cont.).
Biomarker
Marker
Assay type
Specimen
Function
HIV infection
Naive
On cART
NK cell dysfunction (cont.)
CD107a on NK
CD16 on NK
NK cell activity
Flow cytometry‡
NK cell ADCC capacity Flow cytometry
WB/PBMC
WB/PBMC
Elevated/reduced§ Reduced/no normalization
Reduced
No normalization
WB/PBMC
WB/PBMC
WB/PBMC/
plasma
Elevated
Elevated
Elevated
Monocyte dysfunction
CD14 + CD16 ++ monocytes
CD38 on monocytes
TF
Monocyte activation
Immune response to
bacterial products
Flow cytometry
Flow cytometry
Flow cytometry ¶
Normalized
Reduced/no normalization
–
All associations are positive correlations unless otherwise specified.
†
Incubation with specific antigen or peptides and cytokine production measured by enzyme-linked immunospot (ELISpot) or intracellular cytokine staining.
‡
Functional assay requires ex vivo stimulation and intracellular staining.
§
CD107a expression elevated if measured by surface expressioin ex vivo but reduced if measured in response to experimental stimulus.
¶
Cell bound TF measured by flow cytometry. sTF requires an Enzymatic Activity Assay.
ADCC: Antibody-dependent cell cytotoxicity; BMD: Bone mineral density; cART: Combination antiretroviral therapy; CMV: Cytomegalovirus; CRP: C-reactive protein;
CVD: Cardiovascular disease; EndoCAb: Endotoxin core antibody; HBV: Hepatitis B virus; HCV: Hepatitis C virus; IRD: Immune restoration disease;
LBP: Lipopolysaccharide-binding protein; LPS: Lipopolysaccharide; Mtb: Mycobacterium tuberculosis; NHL: Non-Hodgkin’s lymphoma; NK: Natural killer;
PBMC: Peripheral blood mononuclear cell; s: Soluble; TF: Tissue factor; TNFR: TNF receptor; WB: Whole blood.
D‑dimer is a fibrin degradation product,
which has been associated with CVD and
death in the general population and is elevated in untreated HIV-infected patients [69] .
D‑dimer has also been investigated as a marker
for predicting the development of opportunistic disease and mortality in patients receiving
cART [70] . Following initiation of cART, there
was a significant reduction in plasma levels of
D‑dimer [51,69,71] . Increased baseline plasma levels of D‑dimer before initiating cART and elevated levels immediately preceding an event were
associated with the development of an opportunistic infection [50,52] . In the SMART trial,
increased baseline levels of D‑dimer were also
associated with a higher risk of CVD and allcause mortality [72] . The magnitude of change in
D‑dimer levels following interruption of cART
in the SMART study correlated with changes
in HIV RNA [49] , suggesting that viral replication may drive the production of D‑dimer.
D‑dimer levels have also been correlated with
levels of tissue factor (TF) expressed on activated
monocytes, which were associated with both the
level of HIV RNA and markers of microbial
translocation [73] .
Microbial translocation & associated
immune reponse
With the emergence of the hypothesis that
microbial translocation through the GI tract
might be a driving factor for immune activation in HIV-infected patients, soluble markers of
microbial translocation have recently been studied as novel biomarkers in HIV infection [74–76] .
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Biomarkers Med. (2011) 5(2)
Microbial translocation can be measured
in plasma by direct quantification of bacterial
products, including lipopolysaccharide (LPS), a
major component of the Gram-negative bacterial cell wall [3] , bacterial DNA fragments using
PCR-based assays [77] or other microbe-specific
compounds (e.g., peptidoglycan, a component of
the Gram-positive bacterial cell wall) [78] .
In vivo, LPS binds to LPS-binding protein
(LBP), which is expressed at high concentrations
by numerous cell types during the acute phase of
the innate immune response to bacterial infection [79] . The LPS/LBP complex is then bound by
CD14 and the Toll-like receptor 4 on monocytes.
This leads to monocyte activation and release of
sCD14 into the circulation. Therefore, measure­
ment of sCD14 and LBP can also indirectly
quantify the effects of microbial translocation.
„„
Direct measurement of bacterial
products in vivo
The most commonly used method to quantify
LPS in plasma is the limulus amebocyte lysate
assay, which utilizes a series of enzymatic reactions that mimic the coagulation cascade. This
cascade sequence results in the amplification of
the initial stimulus and accounts for the high
sensitivity of the assay (down to 1 pg/ml endotoxin) [80] . Since this assay is so sensitive, contamination during sample preparation and during the conduct of the assay can easily occur.
Furthermore, the enzymatic reactions are critically time and temperature dependent [81] , and
may be affected by numerous inhibitors present
in plasma [82] . These issues all contribute to the
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Biomarkers of immune dysfunction following combination antiretroviral therapy
Association with other biomarkers
Special Report
Association with clinical outcome
Naive
On cART
Naive (HIV+)
On cART (HIV+)
HIV uninfected
–
sCD14
–
–
–
–
–
–
–
–
–
–
HIV RNA, sCD14, CD8 + T‑cell
activation, D‑dimer
–
–
–
–
–
–
–
–
–
–
–
Myocardial
infarction
poor reproducibility of the assay with reported
interassay variability ranging from 25 to
30% [77,83] . Therefore, multiple levels of quality
control need to be in place when utilizing the
assay for clinical studies.
Plasma LPS is elevated in HIV-infected
patients not receiving cART [3,83,84] . Following
cART, LPS levels significantly reduce but do
not return to levels of healthy controls [83–85] .
In HIV-infected patients living in Africa, the
relationship between elevated levels of LPS and
HIV disease progression is less clear. Some studies have reported elevated levels of LPS [85–87]
and sCD14 [85] , while another study has demon­
strated no change in either of these markers over
time in untreated HIV infection [88] . In this latter study, although LPS and sCD14 levels were
not significantly elevated compared with normal
controls, LBP levels were raised and correlated
(albeit weakly) with the increase in proinflammatory cytokines IL‑6 and TNF‑a. It is unknown
why the relationship between LPS and clinical
outcome might be different in this African cohort
to other studies from the USA, but one possible
explanation might be that the higher prevalence
of co-infections may drive immune activation
and, therefore, obscure the associations usually
seen between LPS and disease progression. In
addition, differences in markers of microbial
translocation may also be confounded by ethnic
differences in the patient population [88,89] and
should be taken into account during ana­lysis.
The 16S genomic rDNA can also be quantified
in plasma using PCR-based techniques. This is a
technically challenging assay and persistent DNA
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Ref.
[130,131,142]
[142–144]
[147,149]
[150]
[3,151]
is often detected in negative controls, as a result
of bacterial DNA contamination from the environment and reagents such as Taq poly­merase. It
is also likely that there is a ‘normal’ background
level of 16S rDNA in healthy humans. We, and
others, have demonstrated that 16S rDNA was
increased in untreated HIV-infected patients
and levels were reduced, but not normalized in
patients receiving cART [Kramski M, Purcell DFJ,
Unpublished Data] [77] . Elevated levels of 16S rDNA
have also been associated with higher HIV RNA
and poor CD4 + T‑cell recovery [77] .
Indirect measures of microbial
translocation: endotoxin-specific
immune response
Plasma LBP levels may measure exposure to
LPS more reliably than direct measurement of
LPS [90] . LBP levels are increased in untreated
HIV-infected patients [3] and patients with
AIDS [91] . However, to our knowledge, the
effect of cART on LBP has not been studied
so far. Endotoxin core antibodies (EndoCAb)
are produced by B cells in response to LPS and
can bind to LPS to neutralize its activity [92] .
EndoCAb titers are significantly reduced in
HIV-infected patients compared with healthy
controls [3,54] . Levels of EndoCAb were significantly lower in patients receiving cART compared with untreated patients [54] . Although levels of EndoCAb have been found to be inversely
correlated with LPS [3] , the levels of EndoCAb
may also be affected by B-cell dysfunction in
HIV [93] and, therefore, may not be a realiable
indirect measure of LPS function.
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Plasma levels of sCD14 are measured by
ELISA. Therefore, unlike the LPS and 16S
rDNA assays, which are both technically challenging, quantification of sCD14 is highly reproducible and could potentially be reliably used
in large-scale clinical studies. sCD14 levels are
elevated in untreated HIV-infected patients,
and are elevated in patients with faster progression to AIDS [3,94] . Similar to LPS, cART leads
to a significant reduction in sCD14, but levels
remain significantly elevated compared with
healthy controls [83,95] . Using detailed longitudinal assessment of LPS and sCD14 in patients
on cART together with mathematical modeling,
we recently demonstrated that both markers will
eventually normalize in patients on long-term
cART [83] .
Relationship of markers of microbial
translocation to each other
A direct correlation between all markers for
microbial translocation would be expected;
however, the relationship between LPS and the
immunological response, involving membranebound CD14, sCD14, LBP and EndoCAb, are
complex, and the interaction among these components may differ in patients who are on and off
cART [96,97] . Studies have demonstrated a significant correlation between 16S DNA and LPS [77] ;
LPS and LBP [91] ; and LPS and sCD14 [3,83,91] .
LBP itself also correlates with sCD14 [3] . In addition, 16S rDNA, LPS and sCD14 have all been
demonstrated to correlate with T‑cell activation
(defined as expression of CD38 +HLA-DR+ on
CD8 + T cells) [Lichtfuss GF, Lewin SR, Jaworwski A,
Crowe SM, Unpublished Data] [3,77] .
„„
Clinical relevance of
microbial translocation
The effects of microbial translocation on reconstitution of CD4 + T cells following cART has
been examined in several studies [3,77,83,98,99] .
Some of these studies demonstrated an inverse
association between CD4 + T‑cell increase and
the amount of circulating microbial products as
measured by levels of LPS [3] or 16S rDNA [77] .
We recently examined the relationship between
LPS, sCD14 and long-term CD4 recovery in a
cohort of 96 patients initiating cART, and used
multivariate ana­lysis to determine factors associated with the speed of CD4 + T‑cell recovery to
over 500 cells/µl [83] . Faster restoration to CD4 +
T‑cell counts of over 500 cells/µl was associated
with, in addition to other factors, higher baseline
CD4 + T‑cell counts, lower baseline LPS levels
and paradoxically higher baseline sCD14 levels,
178
Biomarkers Med. (2011) 5(2)
highlighting the complexity of LPS and sCD14
interactions, which can be both pro- and antiinflammatory [100] . In addition, we did not find
that the level of LPS or sCD14 in patients on
cART was associated with the speed of CD4 +
T‑cell recovery [83] .
Increased microbial translocation has also
been associated with several adverse HIV-related
clinical outcomes, including HIV disease progression, dementia, liver disease and mortality. In cART-naive patients, development of an
opportunistic infection was associated with elevated LPS [85] . Higher plasma levels of LPS and
sCD14 were also associated with HIV-associated
dementia in patients who had either failed or
never received cART [91] .
In patients with HIV–hepatitis C virus
(HCV) co-infection, elevated LPS was associated with more rapid progression of liver disease [101] . Patients with an LPS in the upper
quartile (>42 pg/ml) had a 19‑fold greater risk
of liver disease progression (p = 0.018). By contrast, in a small cross-sectional study of patients
with HIV–hepatitis B virus (HBV) co-infection
(n = 55), we found that LPS was not associated
with an increased risk of liver disease (measured
by liver biopsy) [102] . Whether elevated LPS in the
setting of HIV–HCV co-infection is a cause or a
consequence of liver disease remains uncertain,
but it appears that the impact of HCV–HBV
co-infection is different.
Finally, a recent prospective substudy of
patients enrolled in the SMART study demon­
strated that subjects with sCD14 levels in
the highest quartile had a sixfold higher risk
of death than those in the lowest quartile
(95% CI: 2.2–16.1; p = 0.0004), with minimal change after adjustment for inflammatory
markers, CD4 + T‑cell count and HIV RNA [54] .
Immune dysfunction
„„
T cells
HIV not only significantly reduces the number
of CD4 + T cells and causes the expansion of
CD8 + T cells, but also has a profound effect
on T‑cell function. While the total number
of CD4 + T cells is often normalized following
cART, the composition of the T‑cell populations
and their function are not.
In patients receiving cART, naive CD4 + T cells
often do not return to normal levels [103,104] and
the number of activated and senescent cells is elevated [105] . Senescent cells have shortened telomere
length and are defined as CD28-CD57+, which
are both established biomarkers for immuno­
logical aging in a healthy aging population [106] .
future science group
Biomarkers of immune dysfunction following combination antiretroviral therapy
The activating co-receptor, CD28, expressed on
CD4 + and CD8 + T cells, promotes cell survival,
IL‑2 production, metabolic activity and clonal
expansion [107–110] . Therefore, loss of CD28 is
accompanied by pheno­t ypic changes that inhibit
T‑cell function and the accumulation of CD28T cells is a hallmark of immunologic aging [111] .
In untreated HIV-infected patients, an increase
in CD8 + CD28 - T cells and a decrease in
CD4 +CD28 + and CD8 +CD28 + T cells was associated with progression to AIDS [112] , and loss
of CD28 inversely correlated with an increase
in HLA-DR expression on CD4 + T cells [113] .
Patients receiving suppressive cART showed
increased proportions of senescent T cells,
similar to levels found in the elderly (defined as
CD8 + CD57+ CD28-) [105,106] , and higher numbers of CD57+ CD28 - senescent T cells were
associated with lower arterial distensibility [114] .
Recovery of antigen-specific T‑cell responses
can also be impaired or dysregulated following
cART, especially in patients who initiate cART
at low CD4 + T‑cell counts [115,116] . In HIV–HBV
co-infected patients we recently described little
or no recovery in HBV-specific CD8 + T cells
despite HBV-active cART, which successfully
suppressed both HIV and HBV [117] . In HIVinfected patients co-infected with Mycobacterium
tuberculosis, recovery of M. tuberculosis-specific
cells is variable and can be associated with an
excessive T‑cell-mediated immune response
against M. tuberculosis antigens, often leading
to severe clinical disease [118,119] .
In HIV-infected patients, cytomegalovirus
(CMV)-specific T cells account for close to
10% of all antigen-specific CD4 + and CD8 +
T cells [120] , and there has been recent interest
in the role of these cells in immune activation
and non-AIDS events. The number of CMVspecific T cells was significantly higher in HIVinfected patients on cART compared with
un­infected controls [121,122] and in patients naive
to cART [123] . The percentage of CMV-specific
IFN‑g‑producing CD8 + T cells was also positively correlated with carotid intima thickness
in both HIV patients and uninfected control
subjects, suggesting that an increased inflammatory response to CMV may be contributing to
CVD [124] . Interventions that reduce CMV replication, such as valganciclovir, have recently been
demonstrated to reduce CD8 + T‑cell activation
in HIV-infected patients receiving cART [125] .
This novel strategy requires further evaluation
but may have some role in the future to reduce
inflammation and potentially reduce the risk
of CVD.
future science group
Special Report
In conclusion, not all T‑cell subsets recover
equally following cART and this imbalance
may contribute to ongoing immune dysfunction, even in patients with a normal total CD4 +
T‑cell count.
„„
Natural killer cells
Natural killer (NK) cells are traditionally
defined as CD3 -CD56 + cells, and are further
differentiated by the level of expression of
CD56 and whether there is expression of the Fc
receptor, CD16, also known as the receptor for
antibody-dependent cell cytotoxicity. Therefore,
NK cells can be grouped into the smaller regulating CD3 - CD56 bright CD16 -, the larger, predominately cytolytic CD3-CD56dimCD16 + [126]
and the functionally inert CD3 -CD56 -CD16 +
subsets [127] . Activity of NK cells can also be
assessed by expression of HLA-DR as a marker
of activation [128] and CD107a as a marker of
active degranulation [129–131] .
Early studies in treatment-naive HIV-infected
patients demonstrated a significant reduction in
the total number of NK cells compared with
healthy controls [131–135] . However, recent studies have also demonstrated an altered distribution of NK cell subsets, with an increase in
CD3 - CD56 - CD16 + NK cells compared with
uninfected controls [136] . In addition, in treatment-naive HIV-infected patients, NK cells had
reduced expression of activating surface receptors, and reduced cytotoxicity and cytokine
production with little improvement following
cART (reviewed in [137]). Following cART, the
expanded functionally inert CD3-CD56 -CD16 +
NK cells persist [138] and restoration of CD56 +
NK cell numbers occurs, although at a slower
rate than CD4 + T cells [139–141] .
We and others have observed that NK cells
in HIV-infected patients naive to cART express
high levels of CD107a consistent with active
degranulation, a marker for high cytolytic activity [130,131,142] . Following cART, we observed a
reduction of CD107a expression while the coexpression of CD38 and HLA-DR remained
elevated, consistent with persistent NK cell
activation [Lichtfuss GF, Lewin SR, Jaworwski A,
Crowe SM, Unpublished Data] . In addition, expression of CD16 on NK cells was low in patients
with HIV [143] and did not return to normal levels following cART [142,144] , possibly as a direct
result of NK cell activation [144,145] . The relationship between persistent NK cell dysfunction on
cART and long-term clinical outcomes has not
been determined but is an area of interest for
future research.
www.futuremedicine.com
179
Special Report
Lichtfuss, Hoy, Rajasuriar, Kramski, Crowe & Lewin
„„
Monocytes
Monocytes in peripheral blood are characterized
by the combination of CD14 and -16 surface antigen expression. The majority of monocytes express
CD14 without detectable expression of CD16
(CD14++CD16- monocytes). A minor subset representing 5–15% of monocytes expresses CD16 and
variable levels of CD14, and these cells are phenotypically described as CD14 +CD16 ++ [146] . HIV
preferentially infects these CD14 +CD16 ++ monocytes [147,148] . Although this subset was reported
to be expanded in HIV‑infected individuals in the
pre-cART era [149] , our more recent data show that
the CD14 +CD16 ++ monocyte population was not
significantly increased in HIV‑infected individuals
in the setting of cART [147] .
Hallmarks of monocyte activation include
the shedding of CD14, production of IL‑6 and
catalysis of D‑dimer, and have been discussed
in the previous sections. Similar to T‑cell activation markers, monocyte activation continues
for significant periods of time following suppression of HIV RNA with cART. One measure of
monocyte activation, the surface expression of
CD38, declines following cART, although levels
remain significantly higher than in uninfected
controls [150] . Recent evidence suggests that persistence of markers of monocyte activation may
be independent of persistent T‑cell activation. In
the presence of LPS and other Toll-like receptor
ligands, the surface expression of TF (thrombo­
plastin and CD142) is elevated in untreated HIVinfected patients and correlates with the activation
of the monocyte coagulation cascade, eventually
leading to the cleavage of fibrinogen into D‑dimer.
Chronic monocyte activation may directly
contribute to the elevated risk of CVD, as heightened expression of TF has been linked to myocardial infarction and angina in HIV-uninfected
patients [151] . Activated monocytes are crucial cells
in the pathogenesis of athero­sclerosis: they are
the precursors of fat-laden foam cells, which are
the major cells found within the atheromatous
plaque, potentially contributing to the increased
risk of CVD in HIV-infected patients, even
in those with virologic suppression (reviewed
in [152,153]). Studies to determine the precise role
of monocytes in the pathogenesis of non-AIDS
morbidity and mortality in HIV-infected patients
with virologic suppression on cART are ongoing.
Biomarkers & clinical disease in
HIV-infected patients on cART
Biomarkers of inflammation and coagulation
have been most extensively studied regarding
their relationship with overall mortality, CVD
180
Biomarkers Med. (2011) 5(2)
and cardiovascular mortality. There are far fewer
studies examining the relationship between persistent immune dysfunction and inflammation,
and other non-AIDS clinical diseases, including malignancy. There is a clear relationship
between lower total CD4 + T‑cell counts and rates
of malignancy (both HIV related and non-HIV
related) [154] , and the cytokines, IL6, IL‑10 and
sCD30, have all been associated with an increased
risk of non-Hodgkin’s lymphoma [155] .
HIV-infected adults have a higher prevalence
of low bone mineral density (BMD; 40–83%)
compared with HIV-uninfected subjects, and
HIV-infected patients receiving cART have a
lower BMD than HIV-infected patients naive to
cART [156] . Baseline levels of hsCRP and IL‑6,
as well as change in IL‑6, are associated with
lower BMD in the general population [157] , and
also associated with greater risk of fragility fracture [158] . There are currently no data evaluating
levels of inflammatory cytokines or immune activation on the prevalence of osteopenia or osteo­
porosis, or change in BMD in HIV patients;
given the importance of this complication of
cART, further studies are warranted.
Conclusion
Despite control of viral replication and CD4 +
T‑cell reconstitution following cART, HIVinfected patients experience high rates of nonAIDS events, including CVD, malignancy and
premature death. Current research on biomarkers in HIV infection is focusing on the potential
links between persisting immune activation and
dysfunction, and adverse clinical outcomes following cART. While a number of biomarkers
have been evaluated in large clinical prospective
studies, none of these biomarkers are used in routine clinical practise. Further work is still needed
to define the cause and effect of the relationship
between the relevant biomarker and clinical
outcome, and whether reduction of a specific
biomarker following a therapeutic intervention
leads to improved clinical outcomes.
Future perspective
After years of success in the use of cART to manage HIV infection, clinicians now face the challenge of reducing non-AIDS events in patients
on cART. Unfortunately, CD4 + T‑cell count and
HIV RNA as sole biomarkers are unable to fully
predict health outcomes in HIV-infected patients
on cART. The recent clinical trials of IL‑2 clearly
demonstrated that an increase in total CD4+ T‑cell
count did not correlate with improved health outcomes for patients receiving IL‑2 in addition to
future science group
Biomarkers of immune dysfunction following combination antiretroviral therapy
cART [159] . In addition to conventional biomarkers used in HIV-uninfected patients that predict
diseases such as CVD, novel biomarkers that are
highly reproducible will be needed to monitor
ongoing immune activation and dysfunction in
patients on cART. Larger longitudinal clinical
studies are necessary to convincingly demonstrate
clear associations between biomarkers and clinical outcomes. In addition, continuing efforts in
fundamental immunology research are needed
to understand the mechanistic links between
biomarkers and clinical disease. These studies
will also potentially enable the development of
therapeutic interventions beyond cART to reduce
the incidence of non-AIDS events.
Special Report
Financial & competing interests disclosure
GF Lichtfuss is funded by scholarships from Monash
University, the Burnet Institute and The Alfred Hospital.
R Rajasuriar is a recipient of the Kings Scholarship by the
Malaysia government. SM Crowe is a National Health and
Medical Research Council (NHMRC) of Australia
Principal Research Fellow. SR Lewin is a NHMRC practitioner fellow. This work was supported by the NHMRC
and the Alfred Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the
manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
Executive summary
HIV-infected patients receiving combination antiretroviral treatment continue to experience worse health outcomes
compared with the uninfected population
ƒƒ Combination antiretroviral treatment (cART) successfully suppresses HIV RNA and restores the total number of CD4+ T cells to normal
levels in most patients.
ƒƒ HIV-infected patients on long-term cART experience higher rates of cardiovascular disease (CVD), non-AIDS malignancy, neurocognitive
impairment, and liver, kidney and bone disease.
ƒƒ HIV-infected patients on long-term cART have a shorter life expectancy compared with the general population.
cART fails to normalize generalized immune activation of HIV patients
ƒƒ Cellular markers for immune activation (e.g., CD38/HLA-DR coexpression on CD4+ and CD8+ T cells) are elevated in patients on cART
and has been linked to impaired CD4+ T‑cell recovery and increased mortality.
ƒƒ Plasma markers for inflammation (e.g. C-reactive protein, IL-6 and D‑dimer) are elevated in patients on cART, and are associated with
increased CVD, opportunistic disease and all-cause mortality.
ƒƒ HIV is associated with elevated circulating microbial products.
ƒƒ In HIV-infected patients on or off cART, microbial products (e.g., lipopolysaccharide or bacterial 16S rDNA in plasma) are significantly
elevated, and these correlate with markers of T‑cell activation.
ƒƒ Elevated levels of microbial products are associated with the development of opportunistic disease, slower T‑cell recovery and faster
progression of liver disease in patients co-infected with hepatitis C virus.
ƒƒ The immunological response specific for microbial products can be measured (e.g., soluble CD14, lipopolysaccharide-binding protein
or endotoxin core antibody), and in HIV-infected patients receiving cART, elevated soluble CD14 been associated with an increased risk
of mortality.
cART fails to normalize the composition of the T‑cell compartment
ƒƒ Overall, total CD4+ T‑cell numbers return to normal following cART, but there is skewed recovery of some CD4+ T‑cell
subpopulations.
ƒƒ HIV-infected patients receiving cART have increased proportions of CD28-CD57+ T cells, a sign of immunological senescence which is
associated with increased CVD.
ƒƒ The repertoire of antigen-specific T cells is altered in some patients receiving cART and is skewed towards particular common
pathogens (e.g., cytomegalovirus).
cART fails to normalize phenotype & function of other immune cells
ƒƒ Natural killer cells, which are important for antiviral immune response and tumor surveillance, are altered by HIV infection and are
characterized by increased activation and a large functionally inert subpopulation that is only slowly restored following cART.
ƒƒ Monocytes, the source of several plasma markers of immune activation (e.g., sCD14), show increased expression of tissue factor,
which mediates the catalysis of D‑dimer, which can enhance coagulation.
ƒƒ The role of natural killer cells and monocytes in the pathogenesis of disease in cART patients is currently being investigated, and they
are potentially linked to enhanced CVD.
Conclusion
ƒƒ Increased morbidity and mortality in HIV patients receiving cART is associated with incomplete restoration of immune function.
ƒƒ Biomarkers for immune activation and immune dysfunction are useful research tools to understand pathogenesis of and increased risk
for non-AIDS events.
ƒƒ Further research of these biomarkers is required to reliably predict adverse health outcomes and to develop novel interventions to be
used in addition to cART for the management of HIV infection.
future science group
www.futuremedicine.com
181
Special Report
Lichtfuss, Hoy, Rajasuriar, Kramski, Crowe & Lewin
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