Role of R5 phenotypic variation in mother-to-child transmission of HIV-1

chronic viral infections transmitted to infants: from mechanisms to prevention and care Meetin

Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

SummaryHuman embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model

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

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 (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. 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 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. CONCLUSIONS/SIGNIFICANCE: 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 infectio

Recombinant CXCR4/CCR5 hybrid receptors as tools for studies of HIV-1 receptor usage

The chemokine receptors CCR5 and CXCR4 are required, together with CD4, for the entry of HIV-1 into target cells. CCR5 using HIV-1 dominates during transmission and the asymptomatic phase of infection. During progression, virus phenotypes with the ability to use CXCR4 emerge in about 50% of infected individuals. Individuals continuously harbouring CCR5-restricted isolates still progress to AIDS. Differences among CCR5 using isolates, has been found and an evolution towards an altered mode of CCR5 coreceptor use and a reduced sensitivity to inhibition by natural CCR5 ligands has also been described. With the aim to study interactions of natural ligands and HIV-1 isolates with these chemokine receptors, a set of hybrid CXCR4/CCR5 receptors were constructed. Signalling response to their respective natural ligands, SDF-1 and RANTES were studied and prototypic R5 and X4 isolates (HIV-1BaL and HIV-1IIIB) were tested for their ability to use these chimeric receptors. The results showed that ligands and virus use different receptor epitopes which, in turn, vary between the two receptors. Further, the evolution of primary HIV-1 isolates was studied. A total of 246 sequential primary HIV-1 isolates were studied. Using our chimeric CXCR4/CCR5 receptors, we showed that R5 isolates from immunosuppressed individuals are distinct from those isolated from individuals with higher CD4+ T-cell counts, with regards to coreceptor usage. The analysis also showed that the ability to utilize chimeric receptors correlated inversely with the sensitivity to RANTES inhibition of infection. The R5 isolates used receptor chimeras to various degrees. Based on these results, the R5 viruses could be subdivided into two groups: the R5narrow phenotype and the R5broad phenotype. The R5narrow phenotype is defined as viruses that use wt CCR5 but no chimeric receptors, whereas viruses using at least one chimeric receptor in addition to CCR5, are designated R5broad viruses. The mode of coreceptor use by paired plasma and CSF isolates from HIV-1 infected individuals with varying degree of immunodeficiency and neuropathology were studied. The R5 viral phenotypes predominated both in plasma and in CSF. We were able to identify discordant plasma/CSF wt coreceptor use but also, varying R5 viral phenotypes in the paired isolates within individual patients. There were no characteristic patterns of receptor use that could distinguish CSF from plasma isolates. R5 virus use of chimera FC-2 correlated highly with immunosuppression. Efficient chimeric receptor use also correlated, with an increased resistance to inhibition by the CCR5 antagonist TAK-779. In conclusion, our findings propose that alterations in the mode of CCR5 use may be a key event in R5 virus pathogenesis. We believe that R5 virus ability to utilize these CXCR4/CCR5 chimeric receptors reflects a more flexible and more efficient CCR5 usage, which may include a reduced dependency upon interactions with the N-terminal of the receptor for infection. The findings are important, not only with regards to R5-virus pathogenesis and optimization of emerging treatment with CCR5 antagonists, but also for HIV-infection within the CNS

Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.

Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-kappaB, STAT, and AP-1) and hence efficiently funnels several signaling pathways. The assay, performed with the resulting reporter cell line HFF11, has an exceptional high Z-factor and a large signal-to-background ratio. To facilitate cell handling during screening, we introduced a secreted Renilla luciferase as a reporter enzyme. HR36 reporter cells, equipped with the construct, were added to ligands present in a multiwell plate and after addition of coelenterazine they produced a luminescence readout. This procedure economizes cell handling and at the same time increases assay quality and sensitivit

Molecular mapping of epitopes for interaction of HIV-1 as well as natural ligands with the chemokine receptors, CCR5 and CXCR4.

OBJECTIVE: Mapping coreceptor epitopes used by the prototypic R5 and X4 strains, HIV-1BaL and HIV-1IIIB, in comparison with epitopes involved in the activation and signaling induced by the natural ligands, RANTES and SDF-1beta. DESIGN: Receptor hybrids between CCR5 and CXCR4 were constructed. METHODS: Using single-overlap and extension PCR, increasing portions of CCR5 were replaced with corresponding parts of CXCR4. Viral interaction with these constructs was monitored in infection experiments using stably transfected cell lines, and ligand-induced activation of cells transiently expressing the constructs was measured in terms of calcium fluxes. RESULTS: SDF-1beta required an essentially complete CXCR4, whereas RANTES demanded both the N terminus and the first two extracellular loops of CCR5. HIV-1 infection experiments emphasized the importance of the CCR5 N terminus for infection with HIV-1BaL, whereas HIV-1IIIB was less demanding in its use of CXCR4. CONCLUSION: This study, for the first time monitoring CCR5 and CXCR4 ligand activation and HIV-1 interaction concomitantly, indicates that ligands and virus use different receptor epitopes which, in turn, vary between the two receptors. One particular chimera (FC-4b), having its junctional region close to the conserved cysteine in ECL2, functioned as coreceptor for both HIV-1BaL and HIV-1IIIB, but was not activated with RANTES or SDF-1beta. The results provide a basis for tailoring drugs that block viral entry through the two major coreceptors without interfering with their physiological function

Dual R3R5 tropism characterizes cerebrospinal fluid HIV-1 isolates from individuals with high cerebrospinal fluid viral load

Objective: To study the use of major and alternative coreceptors by HIV-1 isolates obtained from paired plasma and cerebrospinal fluid (CSF) samples. Design: Paired plasma and CSF isolates from HIV-1-infected individuals with varying clinical, virologic, and immunologic parameters were assessed for the ability to infect indicator cells expressing a panel of coreceptors with documented expression in the central nervous system (CNS). Methods: HIV-1 isolates obtained from plasma and CSF in 28 individuals with varying viral load, CD4(+) T-cell counts, and with or without AIDS-defining disease were analyzed for the ability to infect NP2.CD4 cells stably expressing a panel of HIV coreceptors (CCR5, CXCR4, CCR3, CXCR6, GPR1, APJ, ChemR23, RDC-1 or BLT1). Results: All isolates from both plasma and CSF utilized CCR5 and/or CXCR4. However, the ability to use both CCR3 and CCR5 (R3R5) was more pronounced in CSF isolates and correlated with high CSF viral load and low CD4(+) T-cell count. Notably, four out of five CSF isolates of subtype C origin exhibited CXCR6 use, which coincided with high CSF viral load despite preserved CD4(+) T-cell counts. The use of other alternative coreceptors was less pronounced. Conclusion: Dual-tropic R3R5 HIV-1 isolates in CSF coincide with high CSF viral load and low CD4(+) T-cell counts. Frequent CXCR6 use by CSF-derived subtype C isolates indicates that subtype-specific differences in coreceptor use may exist that will not be acknowledged when assessing plasma virus isolates. The findings may also bare relevance for HIV-1 replication within the CNS, and consequently, for the neuropathogenesis of AIDS. (C) 2012 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkin

Virus phenotype variability during disease progression of HIV-1 infected children

Background HIV-1 infected children display different clinical evolution, i.e., “fast progression” (FP), “slow progression” (SP) and “long term non progression” (LTNP). One important phenotypic trait linked to disease progression is the evolution of the viral co-receptor use [1], involving a change from CCR5 to CXCR4 use [2]. However, AIDS symptoms can appear in absence of X4 viruses. Recently chimeric receptors between CCR5 and CXCR4 were developed, in which subsequent parts of CCR5 were replaced with corresponding parts of CXCR4 [3]. Their use allowed to document the biological variability of R5 isolates during the pathogenic process in adults [4]. Aim To examine the HIV biological variability in children with different modes of disease progression. Materials and methods 119 isolates from19 HIV-1 positive children were tested for their ability to infect U87.CD4+ cells expressing the wild type receptor CCR5, CXCR4, or one of the 6 chimeric CCR5/CXCR4 receptors. Results Early during infection, all the viruses isolated from 8 SP children used only wild type CCR5 (called R5narrow). In one case, this phenotype persisted during disease progression, whereas in 2 children the virus evolved and was able to use multiple chimeric receptors (called R5broad), and in additional 5 children the virus evolved to CXCR4 usage. Interestengly the FP children, carried close to birth in 2 cases R5narrow virus, in 2 cases R5broad and in one case a dualtropic R5X4 virus. Virus with R5narrow evolved to R5broad in one of the 2 children carrying such phenotype. Both children with R5broad phenotype developed CXCR4 variants during the follow-up. Evolution was observed also in the LTNP, although followed from later on in life (>8 years of age): all tested isolates from 2/6 LTNP remained R5narrow during disease progression; in one child an evolution from R5narrow to R5broad was observed whereas in another child the virus evolved from R5narrow to R5X4. The remaining two children showed R5broad phenotype during the whole followup. Conclusions Our results show that HIV-1 with broad chimeric receptor use is not hampered in transmission, and is more frequent close to birth in FP than in SP children. Viruses from LTNP show a similar phenotypic evolution though at later age