Nonetheless, LT5 plasma was found to provide 50% neutralization with tier-2 and tier-3 Envs (JRFL and PVO.04), indicating that patient LT5 indeed harbored broad neutralizing antibody. Phylogenetic tree of LT-5 sequences (Fig. cellular receptors and coreceptors in the viral entry process, remains exposed on the virus surface under incessant host-selective pressure, particularly that of the autologous neutralizing antibodies. The continual evolution of viral quasispecies by mutation poses an impediment in successful recognition both by virus-specific cellular and humoral immune mechanisms.12,13 In addition, viral sequence diversity also takes complex shape through recombination, particularly in cases of dual infection11,14C19 by different subtypes. Studies of Env diversity would provide information on selective forces such as humoral immunity that might influence the rate of progression of disease and also would help in the identification of determinants on the Env protein that modulate viral response to immune pressure such as neutralizing antibodies. Here, we investigated the genetic properties of genes representing viral quasispecies amplified from plasma of two antiretroviral treatment (ART)-naive slow progressing Indian patients with broadly neutralizing antibody response. First, the neutralization potential of plasma specimens obtained from p32 Inhibitor M36 the two slow progressing patients (NARI-LT1 and NARI-LT5) was examined against 28 Env-pseudotyped viruses comprising tier-1, tier-2, and tier-3 viruses as described previously.20 Plasma samples were diluted in growth media (DMEM supplemented with 5% fetal bovine sera; Invitrogen Inc.) starting from a 1:20 dilution and incubated with Env-pseudotyped viruses for 1?h at 37C. Subsequently 1104 TZM-bl cells21 were added to this mixture in 96-well tray tissue culture plates supplemented with 25?g/ml DEAE Dextran (Sigma Inc.) and further incubated p32 Inhibitor M36 for additional 2 days at 37C in a CO2 incubator. The degree of neutralization of Env-pseudotyped viruses of TZM-bl cells in the presence of LT1 and LT5 plasma was determined by measuring the reduction in relative luminescence units (RLU) as described earlier.20 As shown in Table 1, the majority of viruses tested here were significantly neutralized by LT5 plasma; LT1 plasma also showed substantial neutralization potential p32 Inhibitor M36 albeit to a lesser extent than LT5 plasma. Of viruses, 16/28 showed 50% neutralization in 1:100 LT1 plasma dilutions while 11/28 viruses showed 50% neutralization at 1:500 dilutions and up to 1 1:5361 dilutions. On the other hand, 21/28 viruses showed 50% neutralization to LT5 plasma at 1:100 [including PVO.03 (tier 3 virus) and JRFL (tier 2 p32 Inhibitor M36 virus)], while 12/28 viruses showed 50% neutralization at 1:500 and up to 1 1:8210. Overall, LT5 plasma was found to be a better neutralizer than LT1, although both of them showed broad neutralizing property against the viruses tested here. Table 1. Neutralization Properties of LT1 and LT5 Plasma to Heterologous Env-Pseudotyped Viruses genes amplified directly from plasma of two ART-naive HIV-positive slow progressing individuals. The two individuals were found to possess neutralizing plasma with considerable breadth. Both LT1 and LT5 plasma showed a fair degree of potency but subtype bias toward clade C Envs, especially LT1 plasma. Nonetheless, LT5 plasma was found to provide 50% neutralization with tier-2 and tier-3 Envs (JRFL and PVO.04), indicating that patient LT5 indeed harbored broad Rabbit polyclonal to ZFP161 neutralizing antibody. Phylogenetic tree of LT-5 sequences (Fig. 3) revealed that the Env variants were distinctly separated and clustered with epidemiologically unlinked reference viruses. Additionally, a multiregion hybridization assay (MHAbce v2)24 using LT-5 plasma also indicated recombination events in Env, suggesting that recombinant Env quasispecies constitute a major portion in the plasma (data not shown). It is unclear as to what impact dual infection has on disease progression. Results of previous studies25C30 suggested that superinfection by two different strains tends to result in faster disease progression and it was hypothesized that dual infection facilitates/accelerates viral adaptation and exploitation of cellular niches that would take many years to develop from a homogeneous infecting strain. Interestingly, in our study, we found the presence of distinct subtype B and C strains in the viral quasispecies in plasma in an antiretroviral-naive patient infected for more than 8 years. We also obtained evidence of Env clones from patient LT5 with premature stop codons. This indicated that this particular patient was infected with strains that gave rise to few defective virions. Similar observations were reported by Braibant genes represent viral quasispecies that were in circulation.