Pinter, and J

Pinter, and J. and A370E substitutions in the background of the molecular clone LW_H8res was higher than its affinity for the parental gp120. Surprisingly, no correlation was observed between CD4 binding affinity for monomeric gp120 and the level of neutralization resistance, suggesting that differences in sCD4 neutralization sensitivity between these viruses are only manifested in the context of the tertiary or quaternary structure of gp120 around the viral surface. The results obtained here indicate that this neutralization-sensitive strain IIIB can become neutralization resistant in vivo under selective pressure by neutralizing antibodies but that this resistance may be easily reversed in the absence of immunological pressure. Resistance to antibody neutralization is usually a hallmark of primary human immunodeficiency computer virus type 1 (HIV-1) isolates that have not been passaged through T-cell lines in vitro. This property allows these viruses to persist in vivo and, as such, has significant implications for vaccine strategies aimed at inducing HIV-neutralizing antibodies. Although it is known that HIV-1 becomes neutralization sensitive when cultured in vitro in permanent T-cell lines (9, 21, 42, 48), recent studies have shown that LY 345899 experimental introduction of a chimeric T-cell-line-adapted, neutralization-sensitive simian-human immunodeficiency computer virus in a rhesus macaque resulted in adaptation to a neutralization-resistant phenotype (6, 13, 36). We previously reported that HIV-1 also has the capacity to revert from a neutralization-sensitive to a neutralization-resistant phenotype in vivo. The computer virus variant that was reisolated 7 years after an accidental infection of a laboratory worker in the United States (27, 28, 31, 37, 46), designated FF3346, had a neutralization resistance LY 345899 that was comparable to that generally observed for primary HIV-1 isolates (1). These results suggested that neutralization-sensitive HIV-1 variants lack the capacity to persist in vivo or, at least, lack the capacity to dominate the viral quasispecies in vivo in the presence of neutralizing antibodies. The increased neutralization resistance of isolate FF3346 was accompanied by 38 amino acid substitutions in the gp120 envelope; three of these involved residues in the CD4 binding site (CD4bs) of gp120, A281 (replaced with Val), E370 (replaced with Ala), and K429 (replaced with Glu). E370 is usually important for CD4 binding (24, 25, 39, 40) and contributes up to 57% of the interatomic contacts between gp120 and CD4 (17). We hypothesized that this striking E370A mutation may LY 345899 have been the only substitution allowed in the background of FF3346 that was compatible with replication competence and, at the same time, provided an escape from CD4bs-directed neutralizing antibodies in vivo; somewhat similar escape configurations have been described in vitro (19, 20, 44). As neutralization resistance is likely to be dispensable when neutralizing antibodies are absent from the environment in which the computer virus is replicating, we reasoned that this resistance of isolate FF3346 might be lost during propagation in the absence of neutralizing antibodies, even when only primary lymphocytes, which are typically used for the culturing of primary computer virus isolates, would be used as target cells. Here, we report that in vitro culturing of the neutralization-resistant isolate FF3346 on primary lymphocytes indeed resulted in progeny computer virus with increased sensitivity to neutralization by soluble CD4 (sCD4) and the broadly neutralizing human monoclonal antibody (MAb) b12. This phenotypic change could be attributed primarily to a dual amino acid reversion: an Asn Ser substitution at position 164 in Cetrorelix Acetate the V2 loop and an Ala Glu substitution at position 370 in the C3 region of LY 345899 gp120. The results obtained in this study strongly support the notion that this neutralization resistance of HIV-1 strains is usually a dynamic process that can easily be modulated depending on LY 345899 the milieu of the replicating computer virus (32, 45). MATERIALS AND METHODS PBMCs. For computer virus propagation, experiments were performed with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) with a wild-type CCR5 genotype (CCR5+/+) pooled from at least five healthy blood donors according to previously described procedures (34). The CCR5 genotype was determined by PCR as described elsewhere (12). PBMCs were isolated from buffy coats after Ficoll density gradient centrifugation. For PHA stimulation, 5 106 cells/ml were cultured for 3 days in Iscove’s altered Dulbecco’s medium (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum (HyClone, Logan, Utah), penicillin (10 U/ml), streptomycin (10 g/ml), cyproxin (5 g/ml), and PHA (5 g/ml; Murex Biotech, Dartford, England). Subsequently, cells (106/ml) were produced in the.