Early detection of molecular and biological heterogeneity in HIV-1 isolates from children with slow and fast disease progression.

Francesca Salvatori*, Åsa Björndal°, Fabrizio Mammano, Robert Fredriksson°, Eleonora Tresoldi*, François Clavel, Eva Maria Fenyö°, and Gabriella Scarlatti*.

*Unit of Immunodifferentiation and Immunology of HIV, San Raffaele Scientific Institute, Milan, Italy; °MTC, Karolinska Institute, Stockholm, Sweden; Laboratoire de recherche antivirale, INSERM -U82, Paris, France.

The aim of our study was to identify differences in genetic and phenotype variability according to slow or fast disease progression in children infected from their seropositive mother. Between 2 and 8 sequential primary isolates derived from 9 children with slow or fast HIV-1 disease progression were studied. The biological phenotype of PBMC-derived isolates was determined in U87.CD4 cell lines expressing the chemokine receptors CXCR4, CCR5, CCR3, CCR2, and CCR1. Plasma derived viruses were tested with Tropism Recombination Test (TRT) which relies on homologous recombination of RT-PCR amplified env sequences. HIV-1 V1-C2 env fragments amplified from isolates were screened for diversity by heteroduplex mobility assay (HMA). Sequences were obtained of the env regions V2-C2 and V3. We detected high intra-sample genetic heterogeneity in the V1/V2 and C2 domains of the gp120, as traced by HMA. Two of 4 fast progressors had a heterogeneous viral population within 4 months from birth, suggesting transmission of multiple viral variants from mother to child. Viral heterogeneity increased after 6 months of age in slow progressors. Likewise, we detected phenotype variation over time. With the exception of one child, the first isolate from all children had a R5 phenotype. Later on and independently from the rate of disease progression, X4 viruses emerged in 7 out of 9 cases. Passage of dualtropic isolates in the U87.CD4 cell lines revealed that these were composed of mixtures of monotropic and dualtropic viral variants. Analogously, virus amplified from plasma had similar characteristics. The combined net charge of the amino acid sequences of V2-C2/V3 domains was lower in R5 than X4 monotropic isolates. Interestingly, dualtropic R5X4 viruses showed a R5-like genotype in V2-C2 and a X4 genotype in V3. Early viral heterogeneity may be more frequent than described, and largely dependent on which region of the virus is analyzed. Viral phenotypic variation increased over time, with frequent emergence of X4 monotropic or dualtropic viruses. Interactions between different parts of the envelope molecule are likely to occur, and may influence coreceptor utilization, perhaps in a charge-dependent manner.

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