Increased Sensitivity to Broadly Neutralizing Antibodies
of End-Stage Disease R5 HIV-1 Correlates with Evolution
in Env Glycosylation and Charge
Marie Borggren1., Johanna Repits1., Jasminka Sterjovski2,3, Hannes Uchtenhagen4, Melissa J.
Churchill2,3, Anders Karlsson5, Jan Albert6, Adnane Achour4, Paul R. Gorry2,3,7, Eva Maria Fenyo ¨1,
1Department of Laboratory Medicine, Lund University, Lund, Sweden, 2Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia,
3Department of Medicine, Monash University, Melbourne, Australia, 4Center for Infectious Medicine (CIM), Department of Medicine, Karolinska Institutet, Stockholm,
Sweden, 5Department of Infectious Medicine, South Hospital, Stockholm, Sweden, 6Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet,
Stockholm, Sweden, 7Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia
Background: Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the
objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes
targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing
antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants
that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a
panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset.
Principal Findings: HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to
neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase.
The increased sensitivity correlated with low CD4+T cell count at time of virus isolation and augmented viral infectivity.
Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with
increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced
numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular
changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb).
Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to
the 2G12 epitope.
Conclusions: Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies,
including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of
adaptive molecular Env changes, including loss of glycosylation and gain of positive charge.
Citation: Borggren M, Repits J, Sterjovski J, Uchtenhagen H, Churchill MJ, et al. (2011) Increased Sensitivity to Broadly Neutralizing Antibodies of End-Stage
Disease R5 HIV-1 Correlates with Evolution in Env Glycosylation and Charge. PLoS ONE 6(6): e20135. doi:10.1371/journal.pone.0020135
Editor: Mario A. Ostrowski, University of Toronto, Canada
Received November 3, 2010; Accepted April 26, 2011; Published June 16, 2011
Copyright: ? 2011 Borggren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by grants provided to MJ and AA from the Swedish Research Council, to MJ from the Swedish International Development
Agency/Department for Research Cooperation, to EMF and JA from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant
agreement nu 2014332. Grants were also provided by the Physicians Against AIDS Research Foundation, Clas Groschinskys Foundation, The Royal Physiographic
Society in Lund and Crafoords Foundation. MB and HU are supported by the EC FP6 grant 037611 (EUROPRISE), PRG was supported in part by a grant from the
Australian National Health and Medical Research Council (433915). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Marianne.Jansson@med.lu.se
. These authors contributed equally to this work.
The intra-host evolution of human immunodeficiency virus type
1 (HIV-1) is facilitated by an error-prone reverse transcriptase
(RT) and a high viral turnover . After transmission a population
of distinct but closely related viruses is established and, in constant
interplay with selective forces from the host immune system or
therapeutic agents, the population evolves during the course of the
infection . The viral envelope glycoprotein gp120/gp41
complex (Env) has been shown to exhibit the greatest diversity
among viral proteins .
In the course of the entry process HIV-1 binding via gp120 to
CD4 on the cell surface initiates a series of events including
binding of the coreceptors CCR5 and/or CXCR4 and, ultimately,
gp41-mediated fusion of the viral and cell membranes . CCR5-
restricted (R5) viruses predominate in the early asymptomatic
stages of HIV-1 infection . Viruses able to use CXCR4 instead
of, or in addition to CCR5, for cell entry (X4, or R5X4 viruses,
PLoS ONE | www.plosone.org1 June 2011 | Volume 6 | Issue 6 | e20135
respectively) may emerge later during the disease course and their
appearance has been correlated to accelerated progression to
AIDS [6–8]. However, most infected individuals progress to AIDS
while maintaining an exclusive R5 virus population [7–10]. We
and others have previously studied the evolution of phenotypic
and molecular properties of R5 viruses in patients progressing to
AIDS while maintaining isolates with an exclusive R5 phenotype
[10–20]. In these studies we demonstrated that R5 viruses with
increased fitness, altered receptor interactions and reduced
sensitivity to inhibition by HIV-1 entry inhibitors [10–14,17,19]
may emerge after onset of AIDS. We also described molecular
alterations in the R5 Env, including increased net positive charge
in gp120 along with disease progression .
Since Env is exposed at the viral surface it is also the target for
neutralizing antibodies, which can be detected a few months after
transmission [21,22]. Transmission of the virus from one
individual to another is a bottleneck for virus diversity and the
transmitted viruses have been reported to be relatively sensitive to
neutralization [23,24]. Following development of HIV-1, specific
antibody escape variants will rapidly be selected resulting in
enhanced diversity and a more neutralization-resistant population
[21,25]. However, many primary isolates can still be neutralized
by a few broadly neutralizing antibodies including IgGb12 [26–
28], 2F5 [29–31] and 2G12 [29,32–35]. IgG1b12 recognizes an
epitope that overlaps with the CD4 binding site on gp120 ,
2F5 binds to a conserved linear epitope within the membrane
proximal external region (MPER) of gp41 [30,36] and 2G12
recognizes specific oligomannose glycans on the outer face of
gp120 [35,37,38]. The HIV-1 Env is heavily glycosylated and Env
glycosylation has been suggested to be part of a viral immune
escape strategy [25,39]. Previous studies have also suggested an
enlargement of the Env glycan shield during the immunocompe-
tent phase of the HIV-1 disease [24,40,41].
Despite the relative conserved nature of epitopes targeted by
broadly neutralizing antibodies, mechanisms underlying the
sensitivity of circulating HIV-1 variants to these antibodies are
not fully understood. In this study we have analyzed virus
sensitivity to broadly neutralizing antibodies in relation to Env
modifications, including changes in glycosylation and charge, of
HIV-1 R5 variants evolving during end-stage disease progression.
By the use of a unique panel of R5 isolates obtained sequentially
before and after AIDS onset at severe immunodeficiency we here
reveal that end-stage R5 viruses display increased sensitivity to
neutralization by the TriMab mix of broadly neutralizing
monoclonal antibodies (MAbs) IgGb12, 2F5 and 2G12. Further-
more, we show that increased sensitivity to TriMAb neutralization
correlates with a sharp decline in CD4+T cell count, increase in
viral infectivity and Env with molecular alterations including
reduced numbers of potential N-linked glycosylation sites (PNGS)
and enhanced positive charge. Virus sensitivity to neutralization
by the individual 2G12 MAb was also found to correlate with viral
infectivity and numbers of PNGS and positive charge of Env.
End-stage R5 HIV-1 display increased sensitivity to
broadly neutralizing monoclonal antibodies
In order to explore whether HIV-1 R5 variants evolving during
end-stage disease display altered sensitivity to broadly neutralizing
antibodies, we set out to analyze virus neutralization sensitivity
using a mix of the well characterized and broadly neutralizing
human MAbs 2F5, 2G12 and IgG1b12, known as TriMAb.
Sequentially obtained chronic and end-stage primary R5 isolates
(Table 1) were tested in parallel against TriMab, using a plaque
reduction assay with U87.CD4-CCR5 cells as target cells. R5
isolates from end-stage disease were found to be more sensitive to
neutralization by the TriMAb mix than the corresponding R5
viruses from the chronic phase (Figure 1a). Accordingly, end-stage
AIDS R5 virus from all patients displayed reduced TriMAb IC50
(p=0.028, Figure 1b) in the U87-based neutralization assay.
When TriMAb IC90 was analyzed end-stage R5 virus of three
patients displayed enhanced sensitivity as compared to corre-
sponding chronic stage R5 virus, while both chronic and end-stage
R5 virus of three other patients was not neutralized to 90% at the
highest concentration tested (data not shown). In agreement with
the previously published concordance between the U87-based
neutralization assay and the conventional PBMC-based neutral-
ization assay [42,43], our results on increased TriMab neutrali-
zation sensitivity of end-stage R5 viruses were confirmed when
tested in a PBMC-based neutralization assay (data not shown).
Furthermore, increased sensitivity to TriMAb neutralization
correlated with reduced CD4+T cell count at time of R5 virus
isolation (p,0.001, r=0.84, Table 2). We next tested chronic and
end-stage R5 isolates for sensitivity to the individual 2F5, 2G12
and IgG1b12 MAbs. Several of the patients exhibited R5 virus
from both chronic and end-stage disease that were not neutralized
to 50% by the individual Mabs, even though all viruses could be
neutralized to 50% using the same concentration of TriMAb.
Thus, none of the MAbs could alone significantly distinguish
neutralization sensitivity of virus from chronic and end-stage
disease (Figure 2a–c). Still, end-stage R5 viruses tended to be more
sensitive to 2G12 neutralization since five out of six end-stage R5
viruses were neutralized, in contrast to only two out of six chronic
stage R5 viruses (Figure 2a). Taken together, these findings suggest
that R5 HIV-1 variants with increased sensitivity to broadly
neutralizing antibodies may emerge during severe immunodefi-
Env of end-stage HIV-1 R5 virus variants display reduced
glycosylation and increased positive charge concomitant
with increased sensitivity to TriMab
To examine whether emergence of R5 HIV-1 with increased
sensitivity to broadly neutralizing antibodies was paralleled by Env
evolution during end-stage disease, we analyzed amino acid
modifications that could lead to altered glycosylation pattern.
Numbers of PNGS in 48 env clones derived from the sequentially
obtained primary R5 isolates were analyzed. The average number
of PNGS for each isolate was calculated from the sequences of four
different clones. A significant reduction in numbers of PNGS within
gp160, as well as gp120, was observed when comparing R5 viruses
isolated at end-stage disease with those from the chronic phase
(p=0.028 in both cases; Figure 3a and 3b), while no such clear
pattern was apparent in gp41 (Figures 3c). We also noted that
mutations leading to loss of PNGS mainly clustered in the gp120
variable regions, and in particular in the V2 and V4 regions (Figure
S1). Conversely,the numberof PNGS intheV3 loop wasconserved
between the two time points (Figure S1). Loss of glycosylation in
end-stage Env sequences was further supported by western blot
analysis. Here we noted that the molecular weight of gp160 and
gp120 clones from the end-stage R5 isolates were lower when
compared to clones from corresponding earlier isolate (Figure 4a).
Additionally, end-stage Env clones that were de-glycosylated
following treatment with PNGaseF displayed similar motility as
compared to corresponding PNGaseF treated clones from the
chronic stage (data not shown). Furthermore, length of the gp160
and gp120 amino acid sequences did not appear to differ in a
consistent manner between chronic and end-stage R5 Env clones
(Figure 4b and 4c). Reduced numbers of PNGS in gp160 correlated
R5 HIV Env Changes and Neutralization Sensitivity
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instead with decreasing CD4 counts at time of R5 virus isolation
(Table 2). Since we previously reported on the development of R5
virus variants displaying Env with increased net positive charge in
parallel with increased infectivity [17,18], we analyzed PNGS
numbers in relation to Env net positive charge and viral infectivity.
We here found that reduced PNGS numbers in Env correlated with
increased viral infectivity, assessed as plaque forming units in
U87.CD4-CCR5 cultures, and Env with increased net positive
charge (Table 2). To analyze whether evolution in R5 virus
sensitivity to broadly neutralizing antibodies was associated with the
observed Env modifications, we next assessed virus sensitivity to
found that reduced TriMab IC50 correlated with reduced numbers
of PNGS and increased net positive charge in Env (Table 2). In
relation to viral fitness, we observed that R5 variants with increased
sensitivity to TriMAb also were more infectious in the U87.CD4-
CCR5 cultures (Table 2). Hence, we conclude that R5 HIV-1
variants with increased sensitivity to TriMAb neutralization may
emerge during severe immunodeficiency and display augmented
infectivity, in addition to Env with reduced glycosylation and an
increase in net positive charge.
R5 isolates with increased 2G12 sensitivity appear at low
CD4+T-cell count and display Env modifications
Since end-stage R5 HIV-1 isolates analyzed in this study tended
to be more sensitive to neutralization by the 2G12 MAb than by
IgG1b12 or 2F5, we next compared 2G12 sensitivity with patient
immune status, viral infectivity and Env characteristics. We
established that R5 viruses neutralized by 2G12 (IC50 #25 mg/
ml) were isolated from patients with reduced CD4+T-cell count
(p=0.035; Figure 5a) and displayed increased infectivity when
tested in U87.CD4-CCR5 cultures (p=0.012; Figure 5b). In
addition, the 2G12-sensitive viruses had Envs with reduced
numbers of PNGS and increased positive net charge when
compared to R5 isolates resistant to 2G12 neutralization (IC50
.25 mg/ml) (p=0.015 and p=0.018; Figures 5c and 5d). Since
2G12 binds to a cluster of high mannose glycans [37,38] we
mapped the localization of PNGS modifications in end-stage R5
viruses in relation to the 2G12 epitope. We observed that PNGS
that were previously shown to be essential for 2G12 binding
(N295, N332, N339, N386 and N392) were highly conserved in
gp120 sequences from both 2G12 neutralization-sensitive and -
resistant viruses (Table S1). Molecular models of gp120 were
created based on Env sequences obtained before and after AIDS
onset from patient M (Figure 6). The gp120 models comprised the
core domain as well as the V3, V4 and V5 regions. Glycosylations
were modelled at each PNGS as core saccharides composed of two
N-acetylglycosamine and three mannose residues, found in all N-
linked glycosylations. Comparative analysis of the molecular
models suggested that glycans that were lost in R5 viruses
following AIDS onset were predominantly localized to the outer
solvent accessible domain of gp120 proximal to the 2G12 epitope
(Figure 6). Furthermore the charge of the R5 isolates, calculated
using the molecular models of the gp120 core (that lack mainly
V1/V2), revealed increased positive charge in gp120 of 2G12-
Figure 1. Sensitivity of sequential chronic and end-stage R5 viruses to neutralization by TriMAb. a) Percent TriMAb neutralization of
chronic stage R5 isolates (blue lines) and end-stage R5 isolates (red lines), b) TriMAb IC50 of chronic and end-stage R5 isolates.
Table 1. Patient clinical status, CD4 count and virus
G 1228 260 -9Chronic Asympt. CCR5
+26 End-stage AIDSCCR5
227 Chronic Asympt.CCR5
+6 End-stage AIDSCCR5
230 Chronic Asympt.CCR5
+11 End-stage AIDSCCR5
211 Chronic Asympt.CCR5
+20 End-stage AIDSCCR5
M 668 750
254 Chronic Asympt.CCR5
22 Chronic Asympt.CCR3+CCR5
+16 End-stage AIDS
aPatient code according to .
bCD4+T cells/ml blood at time of virus isolation.
cTime point of virus isolation related to months before and after AIDS diagnosis.
dCoreceptor use determined by infection of U87.CD4 and GHOST(3) coreceptor
indicator cell lines expressing CCR2b, CCR3, CCR5, CXCR4, CXCR6 or BOB .
R5 HIV Env Changes and Neutralization Sensitivity
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sensitive R5 isolates. This corresponded well to the net charge of
gp160 derived from the amino acid sequence (data not shown).
Increased positive surface charge was most prominent in the
vicinity of the 2G12 epitope (Figure S2), when comparing
molecular models of gp120 from chronic and end-stage R5
viruses from patient G, whose end-stage R5 virus also displayed
the greatest gain in 2G12 sensitivity (Figure 5a). Thus, these results
suggest that Env alterations, loss of PNGS and increase in surface
positive charge, in the proximity to the 2G12 epitope, may play
role in 2G12 neutralization.
In the present work, we demonstrate that R5 HIV-1 variants
with increased sensitivity to the TriMAb mixture of broadly
neutralizing IgGb12, 2F5 and 2G12 MAbs may emerge following
AIDS onset at the end-stage of the disease. Increased sensitivity to
TriMAb and to 2G12 alone coincided with enhanced viral
infectivity and Env modifications, including reduced numbers of
PNGS and increased positive charge. Such R5 virus variants
appeared in patients with severe immunodeficiency, as evidenced
by low CD4+T-cell count at time of virus isolation.
Our data suggest that Env binding sites for neutralizing
antibodies, such as the TriMAb mix, are better exposed in R5
viruses emerging in vivo in the absence of appropriate immune
response, in a similar fashion to HIV-1 replicating in the newly
infected host . Indeed, in accordance with findings showing
reduced glycan shield in Env of virus replicating during the acute
infection , we and others , have found that Env of end-stage
R5 virus displayed reduced glycosylation. It is noteworthy to
mention that HIV-1 variants passaged extensively in vitro in the
absence of anti-Env antibodies, i.e. T-cell line adapted viruses
(TCLA), are known to be more sensitive to neutralizing antibodies
as compared to primary isolates [44–46]. Thus, absence of immune
pressure in vivo or in vitro, may result in reversion of escape or
selection of minor virus variants being more sensitive to neutralizing
antibodies, through a mechanism that includes loss of glycans.
Our findings also suggest that, in addition to Env glycan density,
positive net charge of gp120 contributes to R5 virus sensitivity to
neutralizing antibodies. Since sugar residues are negatively
charged, reduction in glycan determinants may also contribute
to elevation of gp120 surface charge. Increase in positive charge
has been reported to have a positive effect on antibody-binding to
the gp120 V3 region . Our previous results disclosed gain in
Env positive charge in other gp120 variable regions of R5 viruses
at end-stage disease , suggesting that Env charge alterations
outside the V3 loop also may influence virus sensitivity to
neutralizing antibodies. Furthermore, end-stage R5 virus displayed
Figure 2. Sensitivity of sequential R5 viruses to neutralization by the 2G12, 2F5 or IgG1b12 monoclonal antibodies. Depicted are a)
2G12, b) 2F5 and c) IgG1b12 IC50 results from the analysis of neutralization sensitivity of sequential chronic and end-stage R5 viruses tested with each
individual monoclonal antibody.
Figure 3. Evolution of PNGS modifications in HIV-1 R5 Env during end-stage disease progression. Differences in numbers of PNGS
within a) gp160, b) gp120 and c) gp41 comparing the average PNGS numbers of four R5 sequences per isolate obtained longitudinally at the
asymptomatic chronic phase and after AIDS onset at end-stage disease.
R5 HIV Env Changes and Neutralization Sensitivity
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increased in vitro fitness, assessed in primary cells and cell lines, as
well as in competition assays [11,17]. Interestingly, increase in
viral infectivity of end-stage R5 viruses also coincides with
reduction in Env PNGS numbers and increased sensitivity to
neutralization by TriMab. In line with these results, Quekkelaar
and colleagues showed that HIV-1 variants sensitive to either
2G12 or 2F5 neutralization displayed increased replicative
capacity . Recently it was also reported that broadly
neutralizing antibodies frequently target a conserved epitope
essential for viral fitness . Thus, evolution of R5 HIV-1 in the
absence of immune selection pressure at end-stage disease may, in
an opportunistic manner, favour viral fitness rather than resistance
to broadly neutralizing antibodies.
Increased sensitivity of end-stage R5 isolates to neutralization by
the IgG1b12 Mab has been reported in previous studies where R5
isolates were obtained cross-sectionally before and after AIDS onset
[12,19]. In contrast, a longitudinal analysis of R5 isolates showed an
increased resistance to IgG1b12 with disease progression . We
could not evaluate neutralization sensitivity using IgG1b12 and 2F5
since half of the analyzed R5 viruses were not neutralized to 50%.
However, we found that 2G12-sensitive R5 isolates were obtained
from patients with lower CD4+T-cell counts than 2G12 resistant
viruses. We also noted that increased 2G12 neutralization sensitivity
correlated with reduced numbers of Env PNGS, which was
surprising since the 2G12 epitope consists of specific oligomannose
glycans positioned on the outer domain of gp120 [35,37,38]. Our
Env sequence analysis revealed that the specific PNGS described to
be important for 2G12 binding in large were conserved, indicating
that the neutralizing effect of 2G12 may not solely be dependent on
these specific glycans. Similarly, it has previously been reported that
HIV-1 variants displaying 2G12 neutralization resistance may
possess all critical PNGS [51,52]. It has also recently been suggested
that nonglycan determinants flanking the CD4 binding site
influenced 2G12 neutralization via a mechanism involving shifts
in the orientation of proximal glycans . Thus, one potential
explanation for our findings could be that the 2G12 epitope is more
exposed if certain surrounding glycans are absent. Indeed,
molecular modeling suggests that Env modifications, including
PNGS loss and positive charge gain, acquired during end-stage
disease development are located in the vicinity of glycans essential
for 2G12 binding.
We believe that knowledge on the natural evolution of HIV-1
sensitivity to broadly neutralizing antibodies, linked to molecular
alterations in the Env structure, may prove important for the
understanding of mechanisms leading to virus escape and
subsequent reversionfromescapeduringHIV-1progressive disease.
Materials and Methods
Patients and virus isolates
HIV-1 isolates were obtained from six patients selected from a
larger cohort of homo- and bisexual men described previously .
This study was approved by the Karolinska Institute Regional
Committee for Research Ethics, ref KI log no. 86:93. Oral
consent was obtained from patients involved, in agreement with
the decision of the Ethics Committee, and this was according to
the ethical standards applied at the time when these isolates were
obtained, between 1987–1995. The selected patients yielded R5
virus isolates throughout the entire course of the disease, including
end-stage AIDS, and in the studied patients the R5 viral
phenotype evolved by gain of enhanced fitness and reduced
sensitivity to RANTES and entry inhibitors along with disease
progression [10,13,17]. The samples were obtained before the
advent of modern combination antiretroviral therapy (cART), but
four of the patients (G, I, J and R) received monotherapy with
zidovudine or didanosine. Isolations were made sequentially, at
the chronic stage when the patients were clinically asymptomatic
and after progression to AIDS at end-stage disease (Table 1).
Figure 4. Molecular weight and peptide backbone length of Env from sequential HIV-1 R5 viruses. a) Mobility in SDS-PAGE gel of gp120
and gp160 clones from R5 HIV-1 isolates as assessed by Western Blot. The results of four Env clones for each R5 isolate obtained during chronic and
end-stage disease from patients H and M are illustrated. Differences in amino acids sequence length of b) gp160 and c) gp120 comparing the average
amino acids length of four R5 Env sequences per isolate obtained longitudinally at the chronic phase and at end-stage disease.
Table 2. Correlations between TriMAb sensitivity, Env PNGS,
Env net charge, virus infectivity and patients CD4+ T cell
PNGS in gp160b
PNGS in gp160b
gp160 net chargeb
p=0.004, R=20.76 p=0.029, R=20.63
p=0.042, R=20.65 p=0.014, R=20.74
CD4+ + T cell countd
p,0.001, R=0.84 p=0.019, R=0.66
aTriMAb sensitivity of R5 isolates, assessed as IC50 mg/ml.
bNumbers of PNGS and net positive charges are the average numbers of four
Env sequences per R5 isolate.
cVirus infectivity evaluated as plaque forming units in U87.CD4-CCR5 cells.
dPatient CD4+T-cell count at time of R5 virus isolation.
R5 HIV Env Changes and Neutralization Sensitivity
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Primary virus isolates were previously obtained by isolation from
peripheral blood mononuclear cells (PBMC) of infected individuals
, as described . Virus stocks were generated by propagation
of isolates in PHA-stimulated (Boule) PBMC from healthy donors.
The R5 phenotype was determined by infection of coreceptor
indicator cell lines GHOST and U87 . Isolates from patient R
(6322 and 8004, see Table 1) displayed the ability to use both
CCR5 and CCR3 in the indicator cell lines. However, since these
isolates did not replicate in PBMC carrying the homozygous
CCR5D32 genotype , they were classified as of R5 phenotype.
HIV-1 neutralization assay
Virus neutralization sensitivity was analysed using the human
MAbs IgG1b12, 2G12, 2F5 and equal molar ratio mixture of the
hree MAbs, known as TriMAb. All MAbs were either purchased
from Polymun Scientific, Vienna, Austria or provided by NIH
AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH. The neutralization assay was setup using the
U87.CD4-CCR5 cell line as previously described [42,43]. In
brief,U87.CD4-CCR5 cells were maintained in Dulbecco’s mod-
ified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf
serum (Thermo Scientific) and antibiotics. One day prior to
infection cells were seeded into 48-well plates in 500 ml of medium,
to obtain a 50% confluent cell layer on the day of infection. At the
day of infection,antibodies and virus stocks weredilutedin infection
medium, i.e.culture mediumcontaining2 mg/ml ofPolybrene. The
antibodies were diluted in four-fold steps, starting from a final
concentration of 25 mg/ml present in the step when antibodies and
virus were preincubated. Dilution of the virus stock was adjusted to
Figure 5. Patient status, viral infectivity and Env characteristics of 2G12 sensitive and resistant R5 isolates. Difference in a) CD4+T cell
count at time of virus isolation, b) viral infectivity, evaluated as plaque forming units in U87.CD4-CCR5 cells, c) gp160 PNGS numbers and d) gp160 net
positive charge of R5 virus isolates being either sensitive (IC50 #25 mg/ml) or resistant (IC50 .25 mg/ml) to 2G12 neutralization. Presented PNGS
numbers and net positive charge represent the average of four Env sequences per R5 isolates. Open circles represent chronic stage viruses, filled
circles represent end-stage viruses.
Figure 6. Models on gp120 localization of lost PNGS in end-stage virus in relation to 2G12 epitope. a) Surface representation of a
molecular model of trimeric gp120 including glycosylation at PNGS, represented as spheres. The trimer is depicted from the orientation of the target
cell with the V3 region facing the viewer. The PNGS that define the core epitope of 2G12 as well as the changes in PNGS from chronic to end-stage R5
viruses (in half or more of the clones) seen in Patient M are indicated as follows; PNGS loss in yellow; gained in red; constant in green; PNGS
composing the 2G12 epitope in blue. The exposed outer face as well as the V3 region of the gp120 monomer are indicated. The lost and gained
PNGS are annotated in yellow and red, respectively. b) Side view of the gp120 trimer with the V3 region pointing downward toward the target cell.
R5 HIV Env Changes and Neutralization Sensitivity
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results of a pretitration plaquetest and a final inoculumof 40 plaque
forming units/well was used. A mixture of 300 ml of diluted
antibodies and 300 ml of diluted virus was preincubated in a
separate 48-well plate for 1 h at 37uC. After the preincubation, the
different antibody-virus mixtures were distributed into triplicate
wells with U87.CD4-CCR5 in a volume of 200 ml well. Control
cultures consisted of wells with cells and virus, but no antibodies.
Two hours after infection 300 ml of infection medium was added to
each well. After overnight incubation, cells were washed with PBS
and 1 ml of infection medium was added to each well. Three days
after infection the cells were washed with PBS and fixed with
methanol-acetone (1:1). To visualize cell nuclei, the fixed cells were
stained with hematoxylin, washed with tap water and dried. The
number of plaques (syncytia) was counted by light microscopy. The
percent neutralization was calculated by determining the reduction
in plaque forming units (p.f.u.)/well in the presence of inhibitory
reagent compared with the control virus cultures containing no
antibodies. The MAb concentrations resulting in 50% of plaque
formation, IC50, was determined.
U87.CD4-CCR5 infectivity assay
The infectivity assay has been described previously [17,43] and
resembles the neutralization assay described above. In brief,
U87.CD4-CCR5 cells were seeded into 48-well plates and
incubated over night to reach 50–60% confluence. Cells were
infected with inoculum virus normalized to a concentration of
functional viral reverse transcriptase (RT) of 8.5 ng RT ml21and
then serially diluted in fivefold step. Functional RT was measured
by the CAVIDI HS kit (Cavidi Tech AB, Uppsala, Sweden). On
day five the cells were fixed and stained as stated above. The
number of p.f.u. per ml was determined. Infectivity was tested for
chronic and end-stage R5 viruses from five out of the six patients,
where results from R5 viruses of patient J were lacking.
Generation of full length env clones and sequence
Full length env clones were generated from genomic DNA as
described previously [11,18]. Briefly, a 2.1 kb env fragment was
amplified by nested PCR and cloned into the pSVIIIenv expression
plasmid. The insert in the pSVIIIenv plasmid was used as template
for sequence analysis of the env gene and from each R5 isolate four
clones were selected according to functionality in a single round
entry assay described previously [12,55]. A set of 7 forward and 8
reverse primers and the ABI prism BigDye Terminator sequencing
kit (Perkin Elmer) were used in the sequencing reaction. The
sequenced segments were assembled to a contig sequence using the
ContigExpress of VectorNTI Advance 10 software (Invitrogen).
Sequences were aligned using ClustalX  followed by manual
editing in GeneDoc [http://www.psc.edu/biomed/genedoc]. The
obtained 48 env sequences were submitted to GenBank and assigned
accession numbers [GenBank:EF600067-EF600114]. For determi-
nation of variation in potential N-linked glycosylation sites (PNGS)
we used the N-glycosite tool in the HIV sequence database [http://
www.hiv.lanl.gov] Maximum likelihood phylogenic trees showed
patient as well as isolate-specific clustering, which argues against
contamination and sample mix-up . We defined the variable
regions of gp120 as follows; V1 (nucleotide 6615–6692 in the HxB2
sequence), V2 (6693–6812), V3 loop (7110–7217), V4 loop (7377–
7478) and V5 (7596–7637).
For analysis of Env expression and proteolytic cleavage, 293T
cells were cotransfected with pSVIIIenv plasmid and pSVTat
plasmids at an 8:1 ratio using Lipofectamine 2000. At 72 hrs after
transfection, cells were resuspended in ice cold lysis buffer (0.5%
[vol/vol] NP-40, 0.5% [wt/vol] sodium deoxycholate, 50 mM
NaCl, 25 mM Tris-HCl [pH 8.0], 10 mM EDTA, 5 mM
benzamidine HCl), and a cocktail of protease inhibitors (Roche)
for 10 min, followed by centrifugation at 130006g for 10 min to
remove cellular debris. Cell lysates were separated in 8.5% (wt/
vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and Env proteins were detected by Western blotting
using rabbit anti-gp120 polyclonal antisera. Env proteins were
visualized using horseradish peroxidase-conjugated antirabbit
immunoglobulin G antibody and enhanced chemi-luminescence
(Promega). To investigate the extent of glycosylation, cell lysates
were incubated with PNGase F (Sigma) according to the
manufacturer’s protocol. Briefly, cleared cell lysate was incubated
over night with 50000 U/ml PNGase (Sigma), at 37uC prior to
SDS-PAGE and Western blotting.
Molecular modelling of gp120 from chronic- and end-
stage R5 viruses
A molecular model of the monomeric HIV-1 gp120 was
generated based on the crystal structure of the HIV-1 gp120 (PDB
ID 2B4C) using the SWISS-MODEL protein modeling server
. The program Coot  was further used for minor localized
adjustments. The trimeric model of HIV-1 gp120 was created
using the recently published model of the CD4-bound gp120
trimer derived from three-dimensional cryo electron tomography
studies . Glycans were modelled at each PNGS, using the
GLYPROT glycan modelling server (http://www.glycosciences.
de/modeling/glyprot/php/main.php), as pentasaccharide struc-
tures common to all N-linked sites composed of two N-
GlcNAc-Man-(Man)2]. The 2G12 epitope was defined for the
modelling as composed of PNGS 295, 332, 339, 386 and 392
[37,38]. Total charges of the molecular models prior to glycan
addition were calculated using the PROPKA server (version 3.0
http://propka.ki.ku.dk/) with standard settings at pH 7.0 [60,61].
Vacuum electrostatic surface potentials were visualized through
calculation of locally averaged surface charges using the protein
contact potential visualization as implemented in Pymol. All
figures were prepared using Pymol (PyMOL Molecular Graphics
System, Version 1.2r1, Schro ¨dinger, LLC).
For statistical analysis we used the Statistica software version 7.
The non-parametric Spearman rank correlation was used for the
analysis of correlations. Comparisons between chronic and end-
stage Env sequences and antibody sensitivity were conducted with
Wilcoxon’s matched pairs test. Non-parametric Mann-Whitney U-
test was used when comparing 2G12 sensitivity to viral and clinical
of HIV-1 R5 emerging during end-stage disease. Local-
ization of PNGS in gp120 of chronic and end-stage R5 virus,
calculated from four sequenced clones per R5 isolate. The
percentage of clones with a PNGS at a given position is color
coded with increasingly darker shades of gray.
Localization of PNGS modifications in gp120
molecular model of the gp120 trimer comparing chronic
Changes in electrostatic surface potential in a
R5 HIV Env Changes and Neutralization Sensitivity
PLoS ONE | www.plosone.org7 June 2011 | Volume 6 | Issue 6 | e20135
and end-stage R5 viruses Visualisation of the electrostatic
surface potential of molecular models of trimeric gp120 from
chronic and end-stage R5 virus of patient G. Positively and
negatively charged parts are shown in blue and red, respectively.
The gp120 trimer is presented from the side with the V3 region
pointing downward toward the target cell, and the approximate
location of the 2G12 core epitope is depicted with arrows.
and 2G12 IC50 for corresponding R5 isolates.
2G12 epitope in sequential R5 Env sequences
The authors are indebted to those providing monoclonal antibodies to
NIH AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH: HIV-1 gp120 MAb IgG1b12 from Dr. Dennis Burton and
Carlos Barbas; HIV-1 gp41 MAb 2F5 and gp120 MAb 2G12 from Dr.
Hermann Katinger. We also thank Dr. Anders Kvist and Dr. Patrik
Medstrand for critical reading of the manuscript.
Conceived and designed the experiments: MB JR MJ. Performed the
experiments: MB JR JS HU MJC. Analyzed the data: MB JR AA EMF
MJ. Contributed reagents/materials/analysis tools: AK JA AA PRG.
Wrote the paper: MB JR HU JA AA PRG EMF MJ. Responsible for
clinical follow-up of patients: AK.
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