Laboratory-adapted HIV-1 Isolates Research Articles

Genetic Signatures of HIV-1 Envelope-mediated Bystander Apoptosis

The envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4(+) T cell loss. However, most of the studies testing this phenomenon have been conducted with laboratory-adapted HIV-1 isolates. This raises the question of whether primary Envs derived from HIV-infected patients are capable of inducing bystander apoptosis and whether specific Env signatures are associated with this phenomenon. We developed a high throughput assay to determine the bystander apoptosis inducing activity of a panel of primary Envs. We tested 38 different Envs for bystander apoptosis, virion infectivity, neutralizing antibody sensitivity, and putative N-linked glycosylation sites along with a comprehensive sequence analysis to determine if specific sequence signatures within the viral Env are associated with bystander apoptosis. Our studies show that primary Envs vary considerably in their bystander apoptosis-inducing potential, a phenomenon that correlates inversely with putative N-linked glycosylation sites and positively with virion infectivity. By use of a novel phylogenetic analysis that avoids subtype bias coupled with structural considerations, we found specific residues like Arg-476 and Asn-425 that were associated with differences in bystander apoptosis induction. A specific role of these residues was also confirmed experimentally. These data demonstrate for the first time the potential of primary R5 Envs to mediate bystander apoptosis in CD4(+) T cells. Furthermore, we identify specific genetic signatures within the Env that may be associated with the bystander apoptosis-inducing phenotype.

Design and Evaluation of Sifuvirtide, a Novel HIV-1 Fusion Inhibitor

Enfuvirtide (T20) is the first and only HIV-1 fusion inhibitor approved for clinical use, but it can easily induce drug resistance limiting its practical application. A novel anti-HIV peptide, termed sifuvirtide, was designed based on the three-dimensional structure of the HIV-1 gp41 fusogenic core conformation. Here we report its in vitro anti-HIV potency, its mechanism of action, as well as the results from Phase Ia clinical studies. We demonstrated that sifuvirtide inhibited HIV-1-mediated cell-cell fusion in a dose-dependent manner and exhibited high potency against infections by a wide range of primary and laboratory-adapted HIV-1 isolates from multiple genotypes with R5 or X4 phenotypes. Notably, sifuvirtide was also highly effective against T20-resistant strains. Unlike T20, sifuvirtide could efficiently block six-helix bundle formation in a dominant negative fashion. These results suggest that sifuvirtide has a different mechanism of action from that of T20. Phase Ia clinical studies of sifuvirtide (FS0101) in 60 healthy individuals demonstrated good safety, tolerability, and pharmacokinetic profiles. A single dose regimen (5, 10, 20, 30, and 40 mg) by subcutaneous injection once daily at abdominal sites was well tolerated without serious adverse events. Pharmacokinetic studies of single and multiple administration of sifuvirtide showed that its decay half-lives were 20.0 +/- 8.6 h and 26.0 +/- 7.9 h, respectively. In summary, sifuvirtide has potential to become an ideal fusion inhibitor for treatment of HIV/AIDS patients, including those with HIV-1 strains resistant to T20.

α-defensins block the early steps of HIV-1 infection: interference with the binding of gp120 to CD4

Abstractα-defensins are antibiotic peptides that act as natural inhibitors of HIV-1 infection. However, the mechanisms of such inhibition are still unclear. Here we demonstrate that α-defensins block the earliest steps in the viral infectious cycle, as documented using an HIV-1 envelope-mediated cell-fusion assay. A broad-spectrum inhibitory activity was observed on primary and laboratory-adapted HIV-1 isolates irrespective of their coreceptor specificity and genetic subtype. A primary mechanism of such inhibition was identified as the ability of α-defensins to bind specifically both to the primary HIV-1 cellular receptor, CD4, and to the viral envelope glycoprotein, gp120. Moreover, treatment of CD4+ T cells with α-defensins caused a dramatic downmodulation of CD4 expression. By monoclonal antibody competition, the regions of interaction with α-defensins were mapped to the D1 domain of CD4 and to a surface contiguous to the CD4- and coreceptor-binding sites of gp120. Consistent with these findings, α-defensins inhibited the binding of gp120 to CD4. These data demonstrate that α-defensins specifically block the initial phase of the HIV infectious cycle and modulate the expression of CD4, a critical receptor in the physiology of T-cell activation.

Characterization of CD4-induced epitopes on the HIV type 1 gp120 envelope glycoprotein recognized by neutralizing human monoclonal antibodies.

The entry of human immunodeficiency virus (HIV-1) into target cells typically requires the sequential binding of the viral exterior envelope glycoprotein, gp120, to CD4 and a chemokine receptor. CD4 binding exposes gp120 epitopes recognized by CD4-induced (CD4i) antibodies, which can block virus binding to the chemokine receptor. We identified three new CD4i antibodies from an HIV-1-infected individual and localized their epitopes. These epitopes include a highly conserved gp120 beta-strand encompassing residues 419-424, which is also important for binding to the CCR5 chemokine receptor. All of the CD4i antibodies inhibited the binding of gp120-CD4 complexes to CCR5. CD4i antibodies and CD4 reciprocally induced each other's binding, suggesting that these ligands recognize a similar gp120 conformation. The CD4i antibodies neutralized laboratory-adapted HIV-1 isolates; primary isolates were more resistant to neutralization by these antibodies. Thus, all known CD4i antibodies recognize a common, conserved gp120 element overlapping the binding site for the CCR5 chemokine receptor.

Candidate microbicides block HIV-1 infection of human immature Langerhans cells within epithelial tissue explants.

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1(Ba-L) infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08-4.77%). HIV-1-infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1-1 microg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1(Ba-L) (an R5 HIV-1 strain) more efficiently infected LC-T cell cocultures when compared with HIV-1(IIIB) (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1.