Coreceptor usage and sensitivity to neutralization of HIV-1 and HIV-2

Sammanfattning: The aim of this project was to study neutralization and coreceptor use in HIV-1 and HIV-2. The first step was to develop a simple and standardized assay for detection and quantification of HIV- 1 neutralizing activity in human sera. The assay is based on plaque formation in the U87 glioma cell line, which is engineered to express CD4 and coreceptors such as CCR5 and CXCR4. Our new assay presents an attractive alternative to PBMC-based assays. In the second paper we developed an additional neutralization assay that uses expression of green fluorescent protein (GFP) in the GHOST(3) cell line as a marker of infection. This assay is a further advance because it is adapted to 96-well microtiter plates and FACS readout. The two assays were equally sensitive and highly reproducible. The assays have comparably high throughput and can therefore be used in HIV vaccine trials and for other large-scale testing. In the third study we used the U87.CD4 and GHOST(3) cell lines to test the evolution of coreceptor usage of 278 sequential HIV-1 isolates from 23 individuals with long and thorough clinical follow-up. Three different patterns of coreceptor usage were identified: i) No evolution of CCR5/CXCR4 use, which was linked to slow disease progression over 8 - 10 years. ii) A change from CCR5 to CXCR4 use, which primarily was observed in patients with progressive disease. iii) Fluctuation between CCR5 and CXCR4 use, which was linked to changes in antiretroviral therapy. Coreceptor use was further dissected using U87.CD4 cell lines expressing chimeric coreceptors, which were constructed by exchanging parts of CCR5 with the corresponding CXCR4 regions. Our data suggest that evolution in coreceptor utilization is a continuous process in which the interaction between the virus envelope and the coreceptor gradually changes. In the forth study, which was carried out to better understand why HIV-2 is less pathogenic than HIV-1, we studied sequential HIV-2 isolates from four HIV-2 infected individuals who displayed different rates of disease progression. Autologous neutralization was tested in a checkerboard fashion using the U87.CD4 neutralization assay. We found that neutralization escape was rare in HIV-2 infection. In addition, we showed, for the first time, that HIV-2 can evolve from CCR5-usage to CXCR4-usage in vivo. Furthermore, the acquisition of CXCR4-usage was closely linked to severe im munodeficiency. Investigation of evolution of the HIV-2 env gene revealed an elongation of the V 1 /V2 domain and changes in glycosylation pattern in patients with slowly progressive disease, which were suggestive of immunological selection according to the "evolving glycan shield" mechanism that recently was proposed for HIV-1. We propose that the V3 domain of HIV-2 has a more open and accessible configuration than that of HIV-1 and that this explains the higher sensitivity to neutralizing antibodies and the broader coreceptor use. In conclusion, we have developed two new cell-line based neutralization assays which may facilitate studies on HIV- 1, HIV-2 and SIV neutralization. We have provided new data on the evolution of coreceptor use in HIV-1 and HIV-2 and a new model of the HIV-2 envelope.