Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

HIV-1 with Multiple CCR5/CXCR4 Chimeric Receptor Use Is Predictive of Immunological Failure in Infected Children

  • Mariangela Cavarelli ,

    cavarelli.mariangela@hsr.it

    Affiliation Viral Evolution and Transmission Unit, DIBIT, Fondazione Centro San Raffaele, Milan, Italy

  • Ingrid Karlsson,

    Affiliation Division of Medical Microbiology/Virology, Department of Laboratory Medicine, Lund University, Lund, Sweden

  • Marisa Zanchetta,

    Affiliation Department of Oncology and Surgical Sciences, Unit of Viral Oncology, AIDS Reference Center, University of Padova, IOV-IRCCS, Padova, Italy

  • Liselotte Antonsson,

    Affiliation Division of Cellular and Molecular Pharmacology, Department of Experimental Medical Science, Lund University, Lund, Sweden

  • Anna Plebani,

    Affiliation Department of Pediatrics, University of Milan, Clinica De Marchi, Milan, Italy

  • Carlo Giaquinto,

    Affiliation Department of Pediatrics, University of Padova, Padova, Italy

  • Eva Maria Fenyö,

    Affiliation Division of Medical Microbiology/Virology, Department of Laboratory Medicine, Lund University, Lund, Sweden

  • Anita De Rossi,

    Affiliation Department of Oncology and Surgical Sciences, Unit of Viral Oncology, AIDS Reference Center, University of Padova, IOV-IRCCS, Padova, Italy

  • Gabriella Scarlatti

    Affiliation Viral Evolution and Transmission Unit, DIBIT, Fondazione Centro San Raffaele, Milan, Italy

HIV-1 with Multiple CCR5/CXCR4 Chimeric Receptor Use Is Predictive of Immunological Failure in Infected Children

  • Mariangela Cavarelli, 
  • Ingrid Karlsson, 
  • Marisa Zanchetta, 
  • Liselotte Antonsson, 
  • Anna Plebani, 
  • Carlo Giaquinto, 
  • Eva Maria Fenyö, 
  • Anita De Rossi, 
  • Gabriella Scarlatti
PLOS
x

Abstract

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 (R5broad 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.

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 R5broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5broad 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 R5broad phenotype, however, the presence of the R5broad 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 R5broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant.

Conclusions/Significance

Our results show that R5broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.

Introduction

Mother-to-child transmission (MTCT) of human immunodeficiency 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 [1].

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 [2]). 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 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 [15]. Evolution of the R5broad phenotype was associated with decreased sensitivity to inhibition by the CC-chemokine RANTES [15]. 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 R5narrow isolates [15]. 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 analysis.

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.

thumbnail
Table 1. Clinical and viral characteristics of HIV-1 infected children(a).

https://doi.org/10.1371/journal.pone.0003292.t001

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 [23]. 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.

thumbnail
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.

https://doi.org/10.1371/journal.pone.0003292.g001

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 [24] (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 [15]. R5X4 viruses used the six chimeric receptors in different combinations, and were not further classified.

Statistical analysis

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 significant.

Results

HIV-1 with R5broad phenotype 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 [3]. Twenty out of 27 children (74.07%) with an R5 virus carried a 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 R5broad virus 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 type CCR5.

The R5broad phenotype 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 R5broad phenotype in the infected newborns was accompanied by a faster progression to immunological 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 R5broad phenotype 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 R5broad isolate 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 virus

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 R5narrow virus had children whose virus displayed the same phenotype. Interestingly, the six mothers carrying R5broad viruses transmitted in all but one case a virus with R5broad phenotype (identical or similar to the mother's virus); in the exceptional mother-child pair, virus with R5narrow phenotype was present in the child. The five mothers with an R5X4 virus transmitted in one case R5X4, in two cases R5broad and in another two cases R5narrow virus. 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 R5broad phenotype 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 transmission

To identify factors predictive of transmission the viral phenotype of 59 mothers (24 transmitting and 35 non transmitting) 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.

thumbnail
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).

https://doi.org/10.1371/journal.pone.0003292.g002

The analysis of the chimeric receptor usage showed that the frequency of the R5narrow phenotype 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 R5broad isolates (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 R5broad viruses from non transmitting mothers can use CCR5 at least as flexibly as viruses from transmitting mothers.

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 R5narrow or R5broad phenotype 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.

Discussion

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 R5broad phenotype) can exist close to infection in the child.

Of utmost relevance are our data, which show that the R5broad phenotype detected in the newborn shortly after birth was predictive of a fast and severe immunological failure. Thus, R5broad viruses 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 [25]. We suggest that pre-existing R5broad viruses may have caused the worsening of the disease. Interestingly, in our study all but one newborn's R5broad virus were capable of FC-4b usage, which indeed was previously shown to be linked to evolution to CXCR4 use in adults [15]. 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 naïve CD4+ cells [26]. The latter predominantly express CXCR4 [26], [27], [28], [29], and are primarily infected by CXCR4 using viruses [30]. It is likely, that the R5broad compared to R5narrow viruses may infect CD4+ naïve cells in addition to memory cells despite the limited expression of the CCR5 molecule, due to their more efficient usage of the coreceptor [15]. Interestingly, Sleasman et al. [27] described that although HIV-1 infected neonates in general contained 10 to 100 fold greater number of infected CD4+ memory cells than naïve cells, those children who rapidly progressed in the disease had high proviral load in the CD4+ naïve cells. Thus, infection of naïve cells by R5broad viruses 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+ naïve 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, R5broad or R5X4. These data lend further support to the lack of restriction in transmission of R5broad viruses and favour the possibility that the maternal viral R5 phenotype is predictive of the transmitted variant.

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 R5broad viruses 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 [34]. It could be envisaged that high levels of CCL3 may be needed to efficiently inhibit R5broad viruses 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 R5broad and R5narrow viruses as well as to study if R5broad viruses have a lower sensitivity to the new CCR5 inhibiting drugs than R5narrow viruses.

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 transmission. 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 infection.

Acknowledgments

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.

Author Contributions

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.

References

  1. 1. UNAIDS (2007) Report on the global AIDS epidemic. Geneve: UNAIDS. UNAIDS2007Report on the global AIDS epidemicGeneveUNAIDS
  2. 2. Scarlatti G (2004) Mother-to-child transmission of HIV-1: advances and controversies of the twentieth centuries. AIDS Rev 6: 67–78.G. Scarlatti2004Mother-to-child transmission of HIV-1: advances and controversies of the twentieth centuries.AIDS Rev66778
  3. 3. Ometto L, Zanchetta M, Mainardi M, De Salvo GL, Garcia-Rodriguez MC, et al. (2000) Co-receptor usage of HIV-1 primary isolates, viral burden, and CCR5 genotype in mother-to-child HIV-1 transmission. AIDS 14: 1721–1729.L. OmettoM. ZanchettaM. MainardiGL De SalvoMC Garcia-Rodriguez2000Co-receptor usage of HIV-1 primary isolates, viral burden, and CCR5 genotype in mother-to-child HIV-1 transmission.AIDS1417211729
  4. 4. Salvatori F, Scarlatti G (2001) HIV type 1 chemokine receptor usage in mother-to-child transmission. AIDS Res Hum Retroviruses 17: 925–935.F. SalvatoriG. Scarlatti2001HIV type 1 chemokine receptor usage in mother-to-child transmission.AIDS Res Hum Retroviruses17925935
  5. 5. Bjorndal A, Deng H, Jansson M, Fiore JR, Colognesi C, et al. (1997) Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol 71: 7478–7487.A. BjorndalH. DengM. JanssonJR FioreC. Colognesi1997Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype.J Virol7174787487
  6. 6. Scarlatti G, Tresoldi E, Bjorndal A, Fredriksson R, Colognesi C, et al. (1997) In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat Med 3: 1259–1265.G. ScarlattiE. TresoldiA. BjorndalR. FredrikssonC. Colognesi1997In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression.Nat Med312591265
  7. 7. Karlsson A, Parsmyr K, Sandstrom E, Fenyo EM, Albert J (1994) MT-2 cell tropism as prognostic marker for disease progression in human immunodeficiency virus type 1 infection. J Clin Microbiol 32: 364–370.A. KarlssonK. ParsmyrE. SandstromEM FenyoJ. Albert1994MT-2 cell tropism as prognostic marker for disease progression in human immunodeficiency virus type 1 infection.J Clin Microbiol32364370
  8. 8. Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, et al. (1993) Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. Ann Intern Med 118: 681–688.M. KootIP KeetAH VosRE de GoedeMT Roos1993Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS.Ann Intern Med118681688
  9. 9. Peters PJ, Sullivan WM, Duenas-Decamp MJ, Bhattacharya J, Ankghuambom C, et al. (2006) Non-macrophage-tropic human immunodeficiency virus type 1 R5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis. J Virol 80: 6324–6332.PJ PetersWM SullivanMJ Duenas-DecampJ. BhattacharyaC. Ankghuambom2006Non-macrophage-tropic human immunodeficiency virus type 1 R5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis.J Virol8063246332
  10. 10. Schuitemaker H, Kootstra NA, de Goede RE, de Wolf F, Miedema F, et al. (1991) Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture. J Virol 65: 356–363.H. SchuitemakerNA KootstraRE de GoedeF. de WolfF. Miedema1991Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture.J Virol65356363
  11. 11. Tuttle DL, Anders CB, Aquino-De Jesus MJ, Poole PP, Lamers SL, et al. (2002) Increased replication of non-syncytium-inducing HIV type 1 isolates in monocyte-derived macrophages is linked to advanced disease in infected children. AIDS Res Hum Retroviruses 18: 353–362.DL TuttleCB AndersMJ Aquino-De JesusPP PooleSL Lamers2002Increased replication of non-syncytium-inducing HIV type 1 isolates in monocyte-derived macrophages is linked to advanced disease in infected children.AIDS Res Hum Retroviruses18353362
  12. 12. Ometto L, Zanchetta M, Cabrelle A, Esposito G, Mainardi M, et al. (1999) Restriction of HIV type 1 infection in macrophages heterozygous for a deletion in the CC-chemokine receptor 5 gene. AIDS Res Hum Retrov 15: 1441–1452.L. OmettoM. ZanchettaA. CabrelleG. EspositoM. Mainardi1999Restriction of HIV type 1 infection in macrophages heterozygous for a deletion in the CC-chemokine receptor 5 gene.AIDS Res Hum Retrov1514411452
  13. 13. Jansson M, Popovic M, Karlsson A, Cocchi F, Rossi P, et al. (1996) Sensitivity to inhibition by beta-chemokines correlates with biological phenotypes of primary HIV-1 isolates. Proc Natl Acad Sci U S A 93: 15382–15387.M. JanssonM. PopovicA. KarlssonF. CocchiP. Rossi1996Sensitivity to inhibition by beta-chemokines correlates with biological phenotypes of primary HIV-1 isolates.Proc Natl Acad Sci U S A931538215387
  14. 14. Koning FA, Kwa D, Boeser-Nunnink B, Dekker J, Vingerhoed J, et al. (2003) Decreasing sensitivity to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization of CC chemokine receptor 5-using, non-syncytium-inducing virus variants in the course of human immunodeficiency virus type 1 infection. J Infect Dis 188: 864–872.FA KoningD. KwaB. Boeser-NunninkJ. DekkerJ. Vingerhoed2003Decreasing sensitivity to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization of CC chemokine receptor 5-using, non-syncytium-inducing virus variants in the course of human immunodeficiency virus type 1 infection.J Infect Dis188864872
  15. 15. Karlsson I, Antonsson L, Shi Y, Oberg M, Karlsson A, et al. (2004) Coevolution of RANTES sensitivity and mode of CCR5 receptor use by human immunodeficiency virus type 1 of the R5 phenotype. J Virol 78: 11807–11815.I. KarlssonL. AntonssonY. ShiM. ObergA. Karlsson2004Coevolution of RANTES sensitivity and mode of CCR5 receptor use by human immunodeficiency virus type 1 of the R5 phenotype.J Virol781180711815
  16. 16. Karlsson I, Antonsson L, Shi Y, Karlsson A, Albert J, et al. (2003) HIV biological variability unveiled: frequent isolations and chimeric receptors reveal unprecedented variation of coreceptor use. AIDS 17: 2561–2569.I. KarlssonL. AntonssonY. ShiA. KarlssonJ. Albert2003HIV biological variability unveiled: frequent isolations and chimeric receptors reveal unprecedented variation of coreceptor use.AIDS1725612569
  17. 17. Scarlatti G, Hodara V, Rossi P, Muggiasca L, Bucceri A, et al. (1993) Transmission of human immunodeficiency virus type 1 (HIV-1) from mother to child correlates with viral phenotype. Virology 197: 624–629.G. ScarlattiV. HodaraP. RossiL. MuggiascaA. Bucceri1993Transmission of human immunodeficiency virus type 1 (HIV-1) from mother to child correlates with viral phenotype.Virology197624629
  18. 18. Scarlatti G, Lombardi V, Plebani A, Principi N, Vegni C, et al. (1991) Polymerase chain reaction, virus isolation and antigen assay in HIV-1-antibody-positive mothers and their children. AIDS 5: 1173–1178.G. ScarlattiV. LombardiA. PlebaniN. PrincipiC. Vegni1991Polymerase chain reaction, virus isolation and antigen assay in HIV-1-antibody-positive mothers and their children.AIDS511731178
  19. 19. Mok JQ, Giaquinto C, De Rossi A, Grosch-Worner I, Ades AE, et al. (1987) Infants born to mothers seropositive for human immunodeficiency virus. Preliminary findings from a multicentre European study. Lancet 1: 1164–1168.JQ MokC. GiaquintoA. De RossiI. Grosch-WornerAE Ades1987Infants born to mothers seropositive for human immunodeficiency virus. Preliminary findings from a multicentre European study.Lancet111641168
  20. 20. Control CfD (1993) 1993 Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Morbid Mortal Wkly Rep 41: 1–18.Control CfD19931993 Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults.Morbid Mortal Wkly Rep41118
  21. 21. Control CfD (1994) 1994 Revised classification system for human immunodeficiency virus infection in children less than 13 years of age. Morbid Mortal Wkly Rep 43: 1–10.Control CfD19941994 Revised classification system for human immunodeficiency virus infection in children less than 13 years of age.Morbid Mortal Wkly Rep43110
  22. 22. De Rossi A, Ades AE, Mammano F, Del Mistro A, Amadori A, et al. (1991) Antigen detection, virus culture, polymerase chain reaction, and in vitro production in the diagnosis of vertically transmitted HIV-1 infection. AIDS 5: 15–20.A. De RossiAE AdesF. MammanoA. Del MistroA. Amadori1991Antigen detection, virus culture, polymerase chain reaction, and in vitro production in the diagnosis of vertically transmitted HIV-1 infection.AIDS51520
  23. 23. Antonsson L, Boketoft A, Garzino-Demo A, Olde B, Owman C (2003) Molecular mapping of epitopes for interaction of HIV-1 as well as natural ligands with the chemokine receptors, CCR5 and CXCR4. AIDS 17: 2571–2579.L. AntonssonA. BoketoftA. Garzino-DemoB. OldeC. Owman2003Molecular mapping of epitopes for interaction of HIV-1 as well as natural ligands with the chemokine receptors, CCR5 and CXCR4.AIDS1725712579
  24. 24. Moore JP, McKeating JA, Weiss RA, Sattentau QJ (1990) Dissociation of gp120 from HIV-1 virions induced by soluble CD4. Science 250: 1139–1142.JP MooreJA McKeatingRA WeissQJ Sattentau1990Dissociation of gp120 from HIV-1 virions induced by soluble CD4.Science25011391142
  25. 25. Casper C, Naver L, Clevestig P, Belfrage E, Leitner T, et al. (2002) Coreceptor change appears after immune deficiency is established in children infected with different HIV-1 subtypes. AIDS Res Hum Retroviruses 18: 343–352.C. CasperL. NaverP. ClevestigE. BelfrageT. Leitner2002Coreceptor change appears after immune deficiency is established in children infected with different HIV-1 subtypes.AIDS Res Hum Retroviruses18343352
  26. 26. Tuttle DL, Coberley CR, Xie X, Kou ZC, Sleasman JW, et al. (2004) Effects of human immunodeficiency virus type 1 infection on CCR5 and CXCR4 coreceptor expression on CD4 T lymphocyte subsets in infants and adolescents. AIDS Res Hum Retroviruses 20: 305–313.DL TuttleCR CoberleyX. XieZC KouJW Sleasman2004Effects of human immunodeficiency virus type 1 infection on CCR5 and CXCR4 coreceptor expression on CD4 T lymphocyte subsets in infants and adolescents.AIDS Res Hum Retroviruses20305313
  27. 27. Sleasman JW, Aleixo LF, Morton A, Skoda-Smith S, Goodenow MM (1996) CD4+ memory T cells are the predominant population of HIV-1-infected lymphocytes in neonates and children. Aids 10: 1477–1484.JW SleasmanLF AleixoA. MortonS. Skoda-SmithMM Goodenow1996CD4+ memory T cells are the predominant population of HIV-1-infected lymphocytes in neonates and children.Aids1014771484
  28. 28. Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR (1997) The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci U S A 94: 1925–1930.CC BleulL. WuJA HoxieTA SpringerCR Mackay1997The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.Proc Natl Acad Sci U S A9419251930
  29. 29. Mo H, Monard S, Pollack H, Ip J, Rochford G, et al. (1998) Expression patterns of the HIV type 1 coreceptors CCR5 and CXCR4 on CD4+ T cells and monocytes from cord and adult blood. AIDS Res Hum Retroviruses 14: 607–617.H. MoS. MonardH. PollackJ. IpG. Rochford1998Expression patterns of the HIV type 1 coreceptors CCR5 and CXCR4 on CD4+ T cells and monocytes from cord and adult blood.AIDS Res Hum Retroviruses14607617
  30. 30. Blaak H, van't Wout AB, Brouwer M, Hooibrink B, Hovenkamp E, et al. (2000) In vivo HIV-1 infection of CD45RA(+)CD4(+) T cells is established primarily by syncytium-inducing variants and correlates with the rate of CD4(+) T cell decline. Proc Natl Acad Sci U S A 97: 1269–1274.H. BlaakAB van't WoutM. BrouwerB. HooibrinkE. Hovenkamp2000In vivo HIV-1 infection of CD45RA(+)CD4(+) T cells is established primarily by syncytium-inducing variants and correlates with the rate of CD4(+) T cell decline.Proc Natl Acad Sci U S A9712691274
  31. 31. Pasquier C, Cayrou C, Blancher A, Tourne-Petheil C, Berrebi A, et al. (1998) Molecular evidence for mother-to-child transmission of multiple variants by analysis of RNA and DNA sequences of human immunodeficiency virus type 1. J Virol 72: 8493–8501.C. PasquierC. CayrouA. BlancherC. Tourne-PetheilA. Berrebi1998Molecular evidence for mother-to-child transmission of multiple variants by analysis of RNA and DNA sequences of human immunodeficiency virus type 1.J Virol7284938501
  32. 32. Wolinsky SM, Wike CM, Korber BT, Hutto C, Parks WP, et al. (1992) Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants. Science 255: 1134–1137.SM WolinskyCM WikeBT KorberC. HuttoWP Parks1992Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants.Science25511341137
  33. 33. Ometto L, Zanotto C, Maccabruni A, Caselli D, Truscia D, et al. (1995) Viral phenotype and host-cell susceptibility to HIV-1 infection as risk factors for mother-to-child HIV-1 transmission. AIDS 9: 427–434.L. OmettoC. ZanottoA. MaccabruniD. CaselliD. Truscia1995Viral phenotype and host-cell susceptibility to HIV-1 infection as risk factors for mother-to-child HIV-1 transmission.AIDS9427434
  34. 34. Meddows-Taylor S, Donninger SL, Paximadis M, Schramm DB, Anthony FS, et al. (2006) Reduced ability of newborns to produce CCL3 is associated with increased susceptibility to perinatal human immunodeficiency virus 1 transmission. J Gen Virol 87: 2055–2065.S. Meddows-TaylorSL DonningerM. PaximadisDB SchrammFS Anthony2006Reduced ability of newborns to produce CCL3 is associated with increased susceptibility to perinatal human immunodeficiency virus 1 transmission.J Gen Virol8720552065