Characterization of minority HIV-1 drug resistant variants in the United Kingdom following the verification of a deep sequencing-based HIV-1 genotyping and tropism assay.

BACKGROUND: The widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, unfortunately, the risk of virologic failure increases with the emergence, and potential transmission, of drug resistant viruses. Detecting and quantifying HIV-1 drug resistance has therefore become the standard of care when designing new antiretroviral regimens. The sensitivity of Sanger sequencing-based HIV-1 genotypic assays is limited by its inability to identify minority members of the quasispecies, i.e., it only detects variants present above ~ 20% of the viral population, thus, failing to detect minority variants below this threshold. It is clear that deep sequencing-based HIV-1 genotyping assays are an important step change towards accurately monitoring HIV-infected individuals. METHODS: We implemented and verified a clinically validated HIV-1 genotyping assay based on deep sequencing (DEEPGEN™) in two clinical laboratories in the United Kingdom: St. George's University Hospitals Healthcare NHS Foundation Trust (London) and at NHS Lothian (Edinburgh), to characterize minority HIV-1 variants in 109 plasma samples from ART-naïve or -experienced individuals. RESULTS: Although subtype B HIV-1 strains were highly prevalent (44%, 48/109), most individuals were infected with non-B subtype viruses (i.e., A1, A2, C, D, F1, G, CRF02_AG, and CRF01_AE). DEEPGEN™ was able to accurately detect drug resistance-associated mutations not identified using standard Sanger sequencing-based tests, which correlated significantly with patient's antiretroviral treatment histories. A higher proportion of minority PI-, NRTI-, and NNRTI-resistance mutations was detected in NHS Lothian patients compared to individuals from St. George's, mainly M46I/L and I50 V (associated with PIs), D67 N, K65R, L74I, M184 V/I, and K219Q (NRTIs), and L100I (NNRTIs). Interestingly, we observed an inverse correlation between intra-patient HIV-1 diversity and CD4+ T cell counts in the NHS Lothian patients. CONCLUSIONS: This is the first study evaluating the transition, training, and implementation of DEEPGEN™ between three clinical laboratories in two different countries. More importantly, we were able to characterize the HIV-1 drug resistance profile (including minority variants), coreceptor tropism, subtyping, and intra-patient viral diversity in patients from the United Kingdom, providing a rigorous foundation for basing clinical decisions on highly sensitive and cost-effective deep sequencing-based HIV-1 genotyping assays in the country.

Synthesis, biological activity, and preliminary pharmacokinetic evaluation of analogues of a phosphosulfomannan angiogenesis inhibitor (PI-88).

The phosphosulfomannan 1 (PI-88) is a mixture of highly sulfated oligosaccharides that is currently undergoing clinical evaluation in cancer patients. As well as its anticancer properties, 1 displays a number of other interesting biological activities. A series of analogues of 1 were synthesized with a single carbon (pentasaccharide) backbone to facilitate structural characterization and interpretation of biological results. In a fashion similar to 1, all compounds were able to inhibit heparanase and to bind tightly to the proangiogenic growth factors FGF-1, FGF-2, and VEGF. The compounds also inhibited the infection of cells and cell-to-cell spread of herpes simplex virus (HSV-1). Preliminary pharmacokinetic data indicated that the compounds displayed different pharmacokinetic behavior compared with 1. Of particular note was the n-octyl derivative, which was cleared 3 times less rapidly than 1 and may provide increased systemic exposure.

Molecular basis for resistance of herpes simplex virus type 1 mutants to the sulfated oligosaccharide inhibitor PI-88.

Herpes simplex virus type 1 variants selected by virus propagation in cultured cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to the presence of PI-88 during their initial infection of cells and/or their cell-to-cell spread. Nucleotide sequence analysis revealed that the deletion of amino acids 33-116 of gC but not lack of gC expression provided the virus with selective advantage to infect cells in the presence of PI-88. Purified gC (Delta33-116) was more resistant to PI-88 than unaltered protein in its binding to cells. Alterations that partly contributed to the virus resistance to PI-88 in its cell-to-cell spread activity were amino acid substitutions Q27R in gD and R770W in gB. These results suggest that PI-88 targets several distinct viral glycoproteins during the course of initial virus infection and cell-to-cell spread.

Chondroitin sulfate characterized by the E-disaccharide unit is a potent inhibitor of herpes simplex virus infectivity and provides the virus binding sites on gro2C cells.

Although cell surface chondroitin sulfate (CS) is regarded as an auxiliary receptor for binding of herpes simplex virus to cells, and purified CS chain types A, B, and C are known to interfere poorly or not at all with the virus infection of cells, we have found that CS type E (CS-E), derived from squid cartilage, exhibited potent antiviral activity. The IC(50) values ranged from 0.06 to 0.2 mug/ml and substantially exceeded the antiviral potency of heparin, the known inhibitor of virus binding to cells. Furthermore, in mutant gro2C cells that express CS but not heparan sulfate, CS-E showed unusually high anti-herpes virus activity with IC(50) values of <1 ng/ml. Enzymatic degradation of CS-E with chondroitinase ABC abolished its antiviral activity. CS-E inhibited the binding to cells of the purified virus attachment protein gC. A direct interaction of gC with immobilized CS-E and inhibition of this binding by CS-E oligosaccharide fragments greater than octasaccharide were demonstrated. Likewise, the gro2C-specific CS chains interfered with the binding of viral gC to these cells and were found to contain a considerable proportion (13%) of the E-disaccharide unit, suggesting that this unit is an essential component of the CS receptor for herpes simplex virus on gro2C cells and that the antiviral activity of CS-E was due to interference with the binding of viral gC to a CS-E-like receptor on the cell surface. Knowledge of the determinants of antiviral properties of CS-E will help in the development of inhibitors of herpes simplex virus infections in humans.