HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children.
ABSTRACT HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5(broad) viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression.
Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5(narrow) phenotype (n = 20), but R5(broad) and R5X4 viruses were also found in seven and one case, respectively. The presence of R5(broad) and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5(broad) phenotype, however, the presence of the R5(broad) virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5(broad) viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5(broad) phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant.
Our results show that R5(broad) viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5(narrow) phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.
- SourceAvailable from: aidsreviews.com[show abstract] [hide abstract]
ABSTRACT: Mother-to-child transmission (MTCT) is the overwhelming source of HIV-1 infection in young children. According to the World Health Organization (WHO), during the year 2003, despite effective antiretroviral (ARV) therapy, there were approximately 700,000 new infections in children worldwide, the majority of whom were from resource-limited countries. Alternative protocols to the long-course and complex regimens of ARV drugs, which in high-income countries have almost eradicated HIV MTCT, have been shown to reduce early transmission rates by 38-50%. However, the accumulation of drug resistance and the long-term toxicities of ARVs mean that alternative approaches need to be developed. Furthermore, transmission via breastfeeding, which accounts for one third of all transmission events, can reduce the benefits of short-course therapies given to women for the prevention of MTCT. The complex mechanisms and determinants of HIV-1 MTCT and its prevention in the different routes of transmission are still not completely understood. Despite the large contribution that many international agencies have made during the past 10-15 years in support of observational and intervention trials, as well as basic scientific research, HIV-1 MTCT intervention trials and basic research often are not integrated, leading to the generation of a fragmented picture. Maternal RNA levels, CD4+ T-cell counts, mode of delivery and gestational age were shown to be independent factors associated with transmission. However, these markers are only partial surrogates and cannot be used as absolute predictors of MTCT of HIV-1. Studies on the role of viral characteristics, immune response and host genomic polymorphisms did not always achieve conclusive results. Although CCR5-using viruses are preferentially carried by HIV-1 infected women as well as transmitted to their infants, the 32-basepair deletion of the CCR5 gene was not shown to influence perinatal MTCT. X4 viruses are apparently hampered in MTCT, although transmission of syncytium-inducing (SI) viruses, which use CXCR4, can occur when the mother carries such virus. Recently, there has been evidence of multiple virus variant transmission during peripartum MTCT. If viral escape from cytotoxic T-lymphocyte (CTL) recognition was repeatedly detected in transmitting mothers, no conclusive results were obtained on the role of the humoral immune response. The hypothesis on the mechanisms of selection during MTCT are still an open question, and include possibly that the transmitted variant is derived from a variant in the mother that escaped immune response, or that transmission is a stochastic event with the random transmission of a limited number of viral variants, or otherwise that selection occurs in the infant through a replication advantage of some transmitted viral variants. Although global access to ARV therapy certainly remains the primary goal to achieve the immediate reduction of MTCT of HIV-1, it is also evident that new and additional innovative strategies are needed.AIDS reviews 6(2):67-78. · 4.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To investigate the relationship between CC chemokine receptor 5 (CCR5) genotype, viral load and co-receptor usage of maternal HIV-1 isolates in perinatal HIV-1 transmission. A total of 181 mothers and infants were studied at the time of delivery. Wild-type (wt) and delta32 CCR5 alleles were determined by means of polymerase chain reaction (PCR). The viral load in maternal plasma samples was determined by a quantitative reverse transcriptase-PCR assay; co-receptor usage of maternal isolates was determined by viral infection in cells stably expressing CCR5 or CXC chemokine receptor 4 (CXCR4) co-receptors. HIV-1 transmission rates in wt/wt and wt/delta32 mothers (14.7 versus 15.8%), and in wt/wt and wt/delta32 infants (14.6 versus 14.3%) were similar. Mothers transmitting infection to wt/delta32 infants had significantly higher HIV-1-RNA levels than those who transmitted infection to wt/wt infants (5.4 versus 4.1 log10 copies/ml, P = 0.03). In wt/wt children there was a positive relationship between transmission rate and maternal viral load over the entire range of HIV-1 values, whereas in wt/delta32 children transmission occurred only at viral loads greater than 4.0 log10 copies/ml. Logistic regression analysis confirmed that the relationship between viral load and transmission varied according to the child's CCR5 genotype (P = 0.035; adjusted for zidovudine prophylaxis and mode of delivery, P = 0.090). Moreover, the majority of wt/wt transmitting mothers had R5-type isolates, whereas none of the wt/delta32 mothers with an R5-type virus transmitted HIV-1 to their wt/delta32 infants. Taken together, these findings suggest that CCR5 delta32 heterozygosity exerts a protective effect against perinatal transmission in children exposed to a low maternal viral burden of an R5-type isolate.AIDS 09/2000; 14(12):1721-9. · 6.41 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.Journal of Virology 11/1997; 71(10):7478-87. · 5.08 Impact Factor
HIV-1 with Multiple CCR5/CXCR4 Chimeric Receptor Use
Is Predictive of Immunological Failure in Infected
Mariangela Cavarelli1*, Ingrid Karlsson2, Marisa Zanchetta3, Liselotte Antonsson4, Anna Plebani5, Carlo
Giaquinto6, Eva Maria Fenyo ¨2, Anita De Rossi3, Gabriella Scarlatti1
1Viral Evolution and Transmission Unit, DIBIT, Fondazione Centro San Raffaele, Milan, Italy, 2Division of Medical Microbiology/Virology, Department of Laboratory
Medicine, Lund University, Lund, Sweden, 3Department of Oncology and Surgical Sciences, Unit of Viral Oncology, AIDS Reference Center, University of Padova, IOV-
IRCCS, Padova, Italy, 4Division of Cellular and Molecular Pharmacology, Department of Experimental Medical Science, Lund University, Lund, Sweden, 5Department of
Pediatrics, University of Milan, Clinica De Marchi, Milan, Italy, 6Department of Pediatrics, University of Padova, Padova, Italy
Background: HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor
use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5broadviruses),
was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation
of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression.
Methodology/Principal Findings: Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and
35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six
different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5narrow
phenotype (n=20), but R5broadand R5X4 viruses were also found in seven and one case, respectively. The presence of
R5broadand R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within
2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5broadphenotype, however, the presence of the
R5broadvirus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the
dualtropic virus, 5 of 6 mothers carrying R5broadviruses transmitted viruses with a similar broad chimeric coreceptor usage.
Thus, the maternal R5broadphenotype was largely preserved during transmission and could be predictive of the phenotype
of the newborn’s viral variant.
Conclusions/Significance: Our results show that R5broadviruses are not hampered in transmission. When transmitted,
immunological failure occurs earlier than in children infected with HIV-1 of R5narrowphenotype. We believe that this finding
is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.
Citation: Cavarelli M, Karlsson I, Zanchetta M, Antonsson L, Plebani A, et al. (2008) HIV-1 with Multiple CCR5/CXCR4 Chimeric Receptor Use Is Predictive of
Immunological Failure in Infected Children. PLoS ONE 3(9): e3292. doi:10.1371/journal.pone.0003292
Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of Medical Sciences and Peking Union Medical College, China
Received May 23, 2008; Accepted September 7, 2008; Published September 29, 2008
Copyright: ? 2008 Cavarelli 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 financial support was received by the Swedish Research Council and by the Istituto Superiore di Sanita ` within the framework of the National
program for AIDS research, Grant no.40G.21, 45G.12 and 40G.56. Only financial support.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Mother-to-child transmission (MTCT) of human immunodefi-
ciency virus type 1 (HIV-1) is the primary mode of infection in
children. In year 2007, an estimated 420.000 new infections
occurred in children aged less than 15 years, most living in Sub-
Saharan Africa .
Several maternal parameters, including advanced clinical stage,
low CD4+ T cell counts, high plasma viral load were associated
with an increased risk of MTCT of HIV-1 (reviewed in ). There
are controversial data concerning the role of viral phenotype in
transmission. Although viruses using CXCR4 as coreceptor (X4
phenotype) can be transmitted when present in the mother,
CCR5-using viruses (R5 phenotype) are the most frequently
detected in newborns [3,4].
Evolution of HIV-1 coreceptor use during disease progression
has been demonstrated in adults as well as children [5,6]. The
evolution usually involves change from CCR5 use (R5 phenotype)
to CXCR4 use alone (X4) or in combination with CCR5 (R5X4)
and/or other minor coreceptors (multitropic viruses). CXCR4-
using viruses can be isolated prior to or during progression to
AIDS, however only from about one-half of patients with overt
AIDS [7,8], thus suggesting that R5 viruses obtained during
clinical progression may differ in phenotypic characteristics from
those obtained during the early stages of infection.
Phenotypic variation characterizes R5 viruses, as demonstrated
by their varying capacity to infect macrophages [9,10,11,12] or
their differential susceptibility to inhibition by CC-chemokines
[6,13,14]. Studies on the entry of R5 viruses into cells expressing
CCR5/CXCR4 chimeric receptors [15,16] showed that the
PLoS ONE | www.plosone.org1 September 2008 | Volume 3 | Issue 9 | e3292
differential susceptibility to inhibition by CC-chemokines depends
on the mode of CCR5 use. In particular, it has been shown that
during disease progression R5 viruses evolve to multiple chimeric
receptor usage (called R5broad), which in turn correlated with
CD4+ T cell decline in the patient . Evolution of the R5broad
phenotype was associated with decreased sensitivity to inhibition
by the CC-chemokine RANTES . The ability of a viral
isolates to use one or more chimeric receptors is most probably a
reflection of a more efficient usage of the CCR5 molecule, as
suggested by our previous results demonstrating a higher
infectivity of the wild-type CCR5 expressing cells with R5broad
compared to R5narrowisolates . In this study we asked the
question whether phenotypic variation, implying different mode of
CCR5 use in pregnant women could play a role in MTCT of
HIV-1 and in turn, also in pediatric disease progression.
Materials and Methods
Patients and virus isolation
Our study population included a total of 59 HIV-1 seropositive
women (24 transmitting and 35 non transmitting) and 28 infected
children. Viral isolates from mother-child pairs were available in
21 cases. Additional seven children were included in the study, but
the maternal samples were not available. Samples from non
transmitting mothers and mother-child pairs were collected within
the framework of two separate cohort studies in Northern Italy.
One cohort consisted of 15 infected mother-child pairs and 17 non
transmitting mothers who took part in a HIV-1 MTCT
multicenter study from 1989 to 1994 [4,6,17,18]. The second
cohort consisted of 9 infected mother-child pairs and 18 non
transmitting mothers collected in the framework of MTCT
multicenter study beginning in 1986 [3,19]. All samples were
from Italian subjects and collected before the introduction of any
MTCT preventive antiretroviral therapy. In 14 out of 16 children
tested the infection was possibly acquired during the intrapartum
period, since negative results were obtained by polymerase chain
reaction (PCR) within the first week of life. Postnatal transmission
was excluded in all cases, as none of the children was breast-fed.
The Ethical Committee approved the use of samples according to
national laws (in 1986, 1991 and 2008). An informed oral consent
was obtained from the pregnant women and, in the case of the
children, from the parents.
The clinical stage of the women and their children (available for
21 transmitting and 24 non transmitting mothers and for 26 out of
28 children respectively), was defined according to the guidelines
of the Centers for Disease Control (CDC) [20,21]. Most of the
mothers were asymptomatic at the time of virus isolation, with the
exception of three mothers: one non transmitting (clinical stage B,
A206) and two transmitting mothers, one with moderate
symptoms (clinical stage B, A225) and one with AIDS (clinical
stage C, A130). CD4+ T cell counts were provided with the
samples in many cases (for 17 transmitting and 28 non
transmitting mothers) [3,17]. HIV-1 p24 antigenemia and plasma
viral load were available for a limited number of women (33 and
17 mothers, respectively) and thus, were not included in the study
HIV-1 infection of the children was determined by virus
isolation and PCR [18,22]. Clinical and immunological data of the
children were obtained throughout follow-up, i.e. until death or for
at least 10 years (Table 1). Children were treated only with mono
or dual antiretroviral therapy (ART). Highly active ART
(HAART) was not administered to children in groups 1, 2 and
3, whereas children in group 4 were treated only after entering
CDC category 3 or C.
Virus isolation was performed from patient’s PBMCs as
previously described [18,22]. HIV-1 p24 antigen (Ag) positive
culture supernatants, collected during first or second culture
passage, were used to prepare virus stocks. Maternal viral isolates
were collected during pregnancy, at delivery or within 5 months
after delivery (Table 2). Virus was isolated from infected children
at an age range between birth and 9 months.
Infection of U87.CD4 cell lines expressing wild type and
CCR5/CXCR4 chimeric receptors
Virus stocks were used to infect human glioma U87.CD4 cells
stably expressing the wild type chemokine receptors CCR5 or
CXCR4, or the six CCR5/CXCR4 chimeric receptors as
previously described [6,16]. Chimeric receptors were obtained
by replacing, beginning from the N-terminal, successively larger
parts of CCR5 with corresponding parts of CXCR4 . In the
resulting chimeras CXCR4 comprised gradually larger parts: the
N-terminal tail only (FC-1), including the first transmembrane
portion (FC-2), the first (FC-4b), second (FC-5 and FC-6) and third
(FC-7) extracellular loops (Figure 1). Parental U87.CD4 cells,
engineered to express CD4 but no chemokine receptor, were used
as negative control.
Cells were infected in duplicate with each virus stock containing
at least 2 ng/ml HIV-1 p24 Ag. The cultures were kept for 7 days
and inspection for syncytia formation was performed at days 1, 3
and 7. Supernatant was collected on day 1, after washing, and at
the last day of infection, and tested for the presence of p24 Ag by
an in-house ELISA assay  (www.aaltobioreagents.ie). Cultures
were only considered for evaluation if HIV-1 p24 value at day 1
was below the lower detection limit of the assay. Viral antigen
production was considered positive when the absorbance at day 7
exceeded 0.2 Optical Density (O.D.).
Viruses able to use only the wild type CCR5 as coreceptor were
defined as R5narrow, whereas R5 viruses able to use the chimeric
receptors, FC-1, FC-2, and FC-4b singularly or in different
combinations were defined as R5broad, according to the previously
published classification . R5X4 viruses used the six chimeric
receptors in different combinations, and were not further
Correlation between R5 phenotype and immunological CDC
stage of infected children were analyzed by Pearson’s chi-square
test. Comparison of the frequencies of the wild type chemokine
receptor and chimeric receptor usage in the two groups of mothers
was done by Fisher’s Exact test and Pearson’s chi-square test. The
Mann-Whitney test and Pearson’s chi-square test were used to
compare the level of CD4+ T cell counts in the two groups of
mothers. Analysis of variance (ANOVA) was performed to
demonstrate the association between CD4+ T cell values and
viral phenotype. Pearson’s chi-square test was used to determine
the correlation between clinical stage and the transmission status
of mothers. Values below 0.05 were regarded as statistically
HIV-1 with R5broadphenotype can be transmitted
Virus isolates obtained close to birth from 28 newborns were
analyzed for phenotypic variability in a cell line expressing wild
type or chimeric chemokine receptors. CXCR4 using virus was
isolated from one newborn only, confirming previous observations
that CCR5 is preferentially used by HIV-1 early after infection
. Twenty out of 27 children (74.07%) with an R5 virus carried a
Pediatric HIV-1 Infection
PLoS ONE | www.plosone.org2September 2008 | Volume 3 | Issue 9 | e3292
virus able to exclusively use wild type CCR5 (R5narrow), whereas
the remaining 7 children (25.93%) harboured virus with broad use
of chimeric receptors (R5broad) (Table 1). These results indicate
that transmission of R5broadvirus occurred in a significant
proportion of children even if the majority of viruses replicating
at a time point close to infection are restricted to the use of wild
The R5broadphenotype is predictive of early
immunological failure in children
Clinical and immunological data obtained during follow-up
were available for 25 out of 27 children carrying an R5 virus close
to birth and for the one child with the R5X4 virus (Table 1). The
presence of viruses with R5broadphenotype in the infected
newborns was accompanied by a faster progression to immuno-
Table 1. Clinical and viral characteristics of HIV-1 infected children(a).
Category of progressionChild code
Age at category diagnosed(c)
CDC 3 CDC BCDC C
CDC 3 or early death ,12 mos B111pBroad (1)44--60
B117p Narrow (5)4-4 11-
B183 Narrow (1.5)---3-
B193Broad (4)66 1228 12
B196 R5X4 (1)66 34 449
B201 Narrow (2)6-69-
B204 Broad (0)66- 384
B314 Broad (0.5)---9-
B380 Narrow (6)99 15-4
CDC3 13–24 mos B252 Broad (1)14- 1430 14
B256Narrow (1.5)17-5 467
CDC3 25–36 mosB32Narrow (3) 2828- 9628
B34p Broad (3)289-- 70
B130Narrow (3) 301 384722
B199 Narrow (2)2724-60 27
CDC3 36–60 mos B3Narrow (6) 65 65-- 54
B115 Narrow (3)5858--59
B136Narrow (3)60--- 64
B145 Narrow (1)48 48 98- 51
B255Narrow (3.5) 46- 12- 12
CDC3 .60 mos or neverB31p Narrow (0)---- 114
B115p Narrow (2.5)----52
B225 Narrow (1)----40
B306Narrow (9)-28-- 72
Lost at follow-up B107pNarrow (1)n.a.n.a.n.a.n.a. n.a.
B139pBroad (4)n.a. n.a.n.a.n.a. n.a.
(a)symbol - means that the event has not occurred. n.a.=not available. Mos means months of age. Age of appearance of the different conditions is always indicated in
(b)Narrow and broad refer to viruses with R5 phenotype. Viruses able to exclusively use wild type CCR5 receptor are defined narrow, whereas those using chimeric
receptors besides the wild type CCR5 are defined broad. In parenthesis is indicated the age in months of the virus phenotype determination.
Statistical analysis: Influence of virus R5broadphenotype on disease progression including children of group 1 and 2, or children of group 1, 2 and 3: p=0.0260 and
p=0.0218 (Pearson’s chi Square), respectively.
(c)Age of entry into clinical or immunological category. Categories are defined according to the Centers for Disease Controls : CDC 3=severe immune suppression;
CDC B=moderate clinical manifestations; CDC C=severe clinical manifestations.
(d)Age of start of mono or dual antiretroviral therapy, not HAART.
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logical failure. Indeed, only children who experienced a severe
decline of the CD4+ T cell counts as fast as within 2 or 3 years of
age or died within 1 year (groups 1, 2 and 3) carried R5broad
viruses close to birth (p=0.026 or p=0.0218, Pearson’s chi-
square, including groups 1 and 2, or groups 1, 2 and 3,
respectively). Whereas none of the children, who were classified
as CDC category 3 after 36 months of age or did not enter this
category during the follow up (groups 4 or 5), had a virus with
R5broadphenotype close to birth. The only child with a R5X4
virus showed also a fast decline of the CD4+ T cell count (classified
CDC 3 within 6 months of age) and died at 44 months.
Specifically, all but one R5broadisolate of the newborns used FC-
4b either alone (n=2) or in combination with FC-2 (n=4),
whereas only one virus isolate used FC-1 and FC-2 (Table 2).
Maternal viral phenotype is predictive of the newborn’s
With the aim to understand if selective processes operate during
transmission, and thus could be predictive for disease progression,
we compared the phenotype of the virus isolates available from 21
mothers close to delivery with that of their newborn child (Table 2).
All ten mothers harbouring an R5narrowvirus had children whose
virus displayed the same phenotype. Interestingly, the six mothers
carrying R5broadviruses transmitted in all but one case a virus with
R5broadphenotype (identical or similar to the mother’s virus); in
the exceptional mother-child pair, virus with R5narrowphenotype
was present in the child. The five mothers with an R5X4 virus
Table 2. Comparison of viral phenotypes of 21 mother-child pairs.
Chimeric receptor usePatient code(a)
Chimeric receptor use
R5- A34p (+3)
R5- R5FC 4b
R5- R5 FC 1-2
R5- R5FC 1-2
R5- R5FC 2
R5- R5FC 4b
R5- R5FC 2
A130 (0)R5-A252 (20.5)R5FC 2
B 136 (+3)
R5-B252 (+1) R5FC 2-4b
R5-A314 (0) R5FC 2-4b
R5-B314 (+0.5)R5 FC 2-4b
B224 (+1) R5-
A225 (0)R5- A31p (0)R5X4FC 1-2-4b-5-6-7
R5- B31p (0)R5 -
R5- A183 (0)R5X4 FC 2-4b-5-6-7
R5- B183 (1.5)R5-
R5- A193 (+4)
R5-R5 FC 2-4b
A196 (0)R5X4FC 4b-5-6-7
B196 (+1)R5X4FC 4b-7
A204 (0)R5X4FC 4b-7
B204 (0)R5FC 2-4b
(a)Isolates from corresponding mother-child pairs are indicated by the same number preceded by a letter: A for mothers and B for children. Time of sampling is indicated
in parenthesis as months before (-), after (+) or within 1 week from (0) delivery/birth. Pairs were grouped according to the mother’s virus phenotype, i.e. first those
carrying an R5narrow, than an R5broadand last an R5X4 virus.
Samples were used to infect U87.CD4 cells expressing wild type CCR5 or CXCR4, or one of the chimeric receptors FC-1, FC-2, FC-4b, FC-5, FC-6 or FC-7. Experiments were
repeated twice. –, means no chimeric receptor is used.
Figure 1. Schematic representation of the chemokine receptors
CCR5 and CXCR4 and the chimeric CCR5/CXCR4 receptors.
Chimeric receptors FC-1, FC-2, FC-4b, FC-5, FC-6 and FC-7 were
obtained by replacing successively larger parts of CCR5 with
corresponding regions of CXCR4.
Pediatric HIV-1 Infection
PLoS ONE | www.plosone.org4September 2008 | Volume 3 | Issue 9 | e3292
transmitted in one case R5X4, in two cases R5broadand in another
two cases R5narrowvirus. Looking at the details, coreceptor use of
child’s virus was narrower than the corresponding mother’s virus
in 7 out of 11 cases, identical in two cases, and a more flexible use
of the CCR5 was observed in one child (pair 252). Thus, a virus
with R5broadphenotype was transmitted from the majority of
mothers carrying such viruses (5 out of 6; 83.3%), indicating that
the maternal viral phenotype is generally preserved during
transmission and can possibly be predictive of the phenotype of
the newborn’s viral variant.
Maternal viral R5 phenotype is not predictive of
To identify factors predictive of transmission the viral
phenotype of 59 mothers (24 transmitting and 35 non transmit-
ting) was investigated. Transmitting mothers carried more often
viruses able to use both CCR5 and CXCR4 as coreceptors than
non transmitting mothers, 29% (7 out of 24) and 8% (3 out of 35),
respectively, but this difference did not reach statistical significance
(p=0.074, Fisher’s Exact Test) (Figure 2). No mother carried a
monotropic X4 virus.
The analysis of the chimeric receptor usage showed that the
frequency of the R5narrowphenotype was similar between the two
groups of mothers, 53% in non transmitting and 65% in
transmitting mothers (17 out of 32 and 11 out of 17, respectively)
(Figure 2). Furthermore, among the R5broadisolates (15 and 6 in
non transmitting and transmitting mothers, respectively), no
difference was observed in the use of FC-1 and FC-2 receptors
between the 2 groups of mothers, while viruses from non
transmitting mothers utilized FC-4b more frequently than viruses
from transmitting mothers (13 out of 15 vs. 2 out of 6, respectively)
with a trend towards significance (p=0.056, Perason’s chi-square
test). This suggests that R5broadviruses from non transmitting
mothers can use CCR5 at least as flexibly as viruses from
Analyses of other factors potentially correlated with risk of
transmission disclosed that the two groups of mothers transmitting
and non transmitting, could not be distinguished by the clinical
stage (p=n.s., Pearson’s chi-square test) and the CD4+ T cell
counts (mean 478 and 426 cells/mm3; p=n.s., Mann-Whitney
test). Furthermore CD4+ T cell values of mothers carrying viruses
with R5narrowor R5broadphenotype did not differ (p=n.s.,
ANOVA), but were significantly different from those of mothers
carrying CXCR4 using viruses (p=0.0014, ANOVA). Thus the
clinical and the immunological stage of the mothers did not seem
to influence the risk of transmission in our cohort.
Biological characteristics of HIV-1 that are critical for the risk of
MTCT continue to be subject to discussion. R5 is the
predominant virus phenotype early in HIV-1 infection, both in
adults and in children born to HIV-1-infected mothers. Since tools
to test the mode of CCR5 use [15,16] have been developed in the
past years, we asked the question whether flexible use of CCR5,
here dissected into narrow and broad phenotype, would influence
transmission of HIV-1 and pediatric disease progression. In the
present study the infected newborns harboured mostly viruses of
R5 phenotype, a significant proportion (25.9%) of these was able
to use chimeric receptors, suggesting that viral variants with a
more flexible and efficient use of CCR5 (the R5broadphenotype)
can exist close to infection in the child.
Of utmost relevance are our data, which show that the R5broad
of a fast and severe immunological failure. Thus, R5broadviruses
seem to determine detrimental effects similar to those known for
CXCR4 using viruses. These data support the finding by Casper et
al, who suggested that the immunological deterioration in HIV-1
infected children precedes the viral phenotypic switch to CXCR4
usage . We suggest that pre-existing R5broadviruses may have
caused the worsening of the disease. Interestingly, in our study all
but one newborn’s R5broadvirus were capable of FC-4b usage,
which indeed was previously shown to be linked to evolution to
CXCR4use in adults . Further studies with sequential follow up
samples from children will clarify if the association with CXCR4
switch occurs also in pediatric HIV-1 infection.
In neonates the memory CD4+ T cells, which express high
levels of CCR5, are 6–7 times less represented than the naı ¨ve
CD4+ cells . The latter predominantly express CXCR4
[26,27,28,29], and are primarily infected by CXCR4 using viruses
. It is likely, that the R5broadcompared to R5narrowviruses may
infect CD4+ naı ¨ve cells in addition to memory cells despite the
limited expression of the CCR5 molecule, due to their more
efficient usage of the coreceptor . Interestingly, Sleasman et al.
 described that although HIV-1 infected neonates in general
contained 10 to 100 fold greater number of infected CD4+
memory cells than naı ¨ve cells, those children who rapidly
progressed in the disease had high proviral load in the CD4+
naı ¨ve cells. Thus, infection of naı ¨ve cells by R5broadviruses may
interfere with CD4+ T cells production, and thus account for the
rapid disease progression observed in children harbouring these
viruses. It remains to be solved why CXCR4 using viruses are not
preferentially maintained during transmission, despite the high
prevalence of CXCR4+ naı ¨ve cells in neonates.
The detailed analysis of the mother’s viruses in comparison to
those of the corresponding child allowed us once again to pinpoint
that the R5 phenotype, either narrow or broad, was usually
maintained during the transmission event. On the contrary, the
R5X4 phenotype was predominantly lost during transmission.
Specifically, mothers with an R5X4 virus transmitted virus with a
whole array of phenotypes, i.e. R5narrow, R5broador R5X4. These
data lend further support to the lack of restriction in transmission
of R5broadviruses and favour the possibility that the maternal viral
R5 phenotype is predictive of the transmitted variant.
Figure 2. Distribution of the viral phenotype of transmitting
and non transmitting mothers. Distribution of R5 (black) vs. R5X4
(white) viruses within all virus phenotypes (n=59 viruses; p=n.s.,
Fisher’s Exact Test) and of R5narrow(dark gray) vs. R5broad(light gray)
within the R5 phenotype (n=49 viruses; p=n.s., Fisher’s Exact Test).
Pediatric HIV-1 Infection
PLoS ONE | www.plosone.org5September 2008 | Volume 3 | Issue 9 | e3292
Our data suggest that mothers carrying R5X4 viruses have
phenotypically highly heterogenous populations. Whether a
selective process or simply a random event governs transmission
remains, however, a topic of discussion. [17,31,32,33].
We showed that mothers harbouring R5broadviruses were not at
higher risk of transmission and the presence of HIV-1 with R5broad
phenotype was not predictive for MTCT of HIV-1. The use of
chimeric receptors suggests a more flexible usage of CCR5, which
in turn may indicate increased viral resistance to inhibition by CC-
chemokines. In this respect, the trend of the non transmitting
mothers to more often carry viruses with the specific usage of the
FC-4b chimeric receptor than transmitting mothers, is intriguing.
Recently, Meddows-Taylor et al. described that non transmitting
mothers have significantly higher levels of CCL3 in the plasma
than transmitting mothers . It could be envisaged that high
levels of CCL3 may be needed to efficiently inhibit R5broadviruses
and, as a consequence, to prevent MTCT of HIV-1. In this
respect, it will be relevant to compare the level of CC-chemokines
in mothers harbouring R5broadand R5narrowviruses as well as to
study if R5broadviruses have a lower sensitivity to the new CCR5
inhibiting drugs than R5narrowviruses.
In summary our data show that approximately one forth of the
newborn’s R5 viruses have the capacity to use CCR5/CXCR4
chimeric receptors indicating that phenotypes with increased
flexibility of co-receptor use are not hampered during transmis-
sion. Conversely these viral variants are significantly linked with
severe immunological failure within the first years of age of the
infected children. These data may have important implications for
timely and appropriate therapeutic choice in pediatric HIV-1
The samples were provided by the National Care System for HIV-1
infected mothers, the Seventh Department of Gynecology and Obstetrics,
and the First and Fourth Department of Pediatrics of the University of
Milan, Italy and by the AIDS Reference Center, Unit of Viral Oncology,
University of Padova, Italy. The authors thank the Italian Register for HIV
Infection in Children for providing clinical data of the children.
The statistical analysis was kindly performed by Clelia Di Serio and
Alessandro Ambrosi from the University Centre of Statistics for Biomedical
Sciences, Vita Salute San Raffaele University, Milan, Italy.
Conceived and designed the experiments: GS. Performed the experiments:
MC IK. Analyzed the data: MC GS. Wrote the paper: MC EMF ADR
GS. Performed testing with the U87 cell lines of some of the children early
isolates and contributed with her expertise on the U87 cell lines expressing
chimeric receptors: IK. Performed the blood sample preparation and virus
isolation of part of the samples: MZ. Has produced the U87 cells
expressing the chimeric receptors and provided the cells as well as the
know-how to the study: LA. Provided part of the samples and clinical data
of mothers and children: AP CG. Provided part of the samples: ADR.
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