Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120

110

135

Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B /C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC 50 ) and IC 90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

Forsman

Beirnaert

Aasa-Chapman

et al. 2008

110

135

“…Changes in the HR1 domain of the gp41 subunit, which comprises the binding site for the fusion inhibitor enfuvirtide, can affect virus sensitivity to this newly approved drug both in vitro and in vivo (46,54). Alternatively, changes in gp120 that affect coreceptor binding can also affect enfuvirtide sensitivity even though this compound binds to gp41 (17,18,42). We have found that reduced coreceptor binding efficiency can slow fusion kinetics, leading to prolonged exposure of the enfuvirtide binding site and increased sensitivity to this fusion inhibitor.…”

Section: Discussionmentioning

confidence: 99%

Impact of Mutations in the Coreceptor Binding Site on Human Immunodeficiency Virus Type 1 Fusion, Infection, and Entry Inhibitor Sensitivity

Reeves

Miamidian

Biscone

et al. 2004

106

111

An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet ␤21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.

“…It is noteworthy, however, that in cases where ENF resistance mutations produced only suboptimal resistance, Env replicative capacity was usually maintained, suggesting that the impact of resistance mutations on viral resistance and viral fitness was not tightly linked (see below). The identification of viral sequences that determine whether a given context is favorable or unfavorable for the expression of high resistance by ENF resistance mutations may prove to be a difficult task, given that multiple discontinuous envelope domains participate in the entry process and can modulate phenotypic resistance to ENF (8,15,34).…”

Section: Vol 80 2006 Evolution Of Hiv-1 Resistance To Enf 8813mentioning

confidence: 99%

“…However, we along with others have shown that baseline susceptibility to ENF is quite variable among viruses from different patients (9,20,39), and variations in domains of Env other than HR1 are thought to account for these differences in baseline susceptibility (8,15,34). In particular, susceptibility to fusion inhibitors appears to be dependent on the affinity of gp120 for the coreceptors as well as on coreceptor density at the cell surface, both of which factors affect fusion kinetics (34).…”

mentioning

confidence: 99%

Role of the Envelope Genetic Context in the Development of Enfuvirtide Resistance in Human Immunodeficiency Virus Type 1-Infected Patients

Labrosse

Morand‐Joubert

Goubard

et al. 2006

Acquired human immunodeficiency virus type 1(HIV-1) resistance to the fusion inhibitor enfuvirtide (ENF)is primarily associated with mutations within the highly conserved first heptad repeat (HR1) region of gp41. Viral env sequences, however, are remarkably variable, and the envelope genetic background could have an important impact on optimal expression of HR1 mutations. We have examined the genetic evolution of env sequences, ENF susceptibility, and Env replicative capacity in patients failing ENF treatment. Sequential plasma-derived virus populations, obtained from six patients initiating ENF treatment as part of a salvage therapy, were studied using a recombinant phenotypic assay evaluating the entire gp120 and the gp41 ectodomains. Regardless of major differences in the baseline ENF susceptibilities, viral populations with similar phenotypic ENF resistance (50% inhibitory concentration, >3,000 ng/ml) were selected under treatment in four of six patients. As expected, in all patients ENF-resistant viruses harbored one or more HR1 mutations (positions 36, 38, and 43). Interestingly, in five patients the emergence of resistance mutations was not associated with reduced Env replicative capacity. Phylogenetic analysis of env sequences in sequential samples from two patients showed that the HR1 mutations had emerged in the context of env quasi-species that were different from those prevalent at baseline. Thus, the envelope genetic context appears to play a critical role in the selection of HR1 mutations and the expression of ENF resistance, thereby conditioning the evolution of HIV-1 under fusion inhibitor selective pressure.Considerable effort is currently being devoted to the development of antiviral agents able to prevent human immunodeficiency virus type 1 (HIV-1) entry into target cells. The HIV-1 entry process is mediated by the trimeric viral envelope glycoproteins (Env) exposed at the surface of the virion (45). The HIV-1 entry is a multistep process (11,14,45). The sequential interaction of the surface subunit, gp120, with CD4 and a chemokine receptor (CCR5 or CXCR4) exposed on the cell membrane triggers conformational changes in the ectodomain of the transmembrane subunit, gp41, which ultimately lead to fusion between the viral and host cell membranes. Like most of the retroviral transmembrane proteins, the ectodomain of gp41 contains an N-terminal fusion peptide followed by two heptad repeat domains (HR1 and HR2), which are connected by a non-helical loop region of 25 to 30 amino acids. Membrane fusion is currently thought to result from the insertion of the fusion peptide into the cellular membrane, and the formation of a six-helix bundle in which the central trimeric HR1 coiled-coil forms three hydrophobic grooves onto which three HR2 domains pack in reverse orientation (4,5,40,43).Although several HIV-1 entry inhibitors have been evaluated in clinical trials and have shown promising prospects for therapy (1, 28), the only entry inhibitor licensed to date is the fusion inhibitor enfuvirtide (ENF; als...