Tropism-independent protection of macaques against vaginal transmission of three SHIVs by the HIV-1 fusion inhibitor T-1249.

We have assessed the potential of the fusion inhibitory peptide T-1249 for development as a vaginal microbicide to prevent HIV-1 sexual transmission. When formulated as a simple gel, T-1249 provided dose-dependent protection to macaques against high-dose challenge with three different SHIVs that used either CCR5 or CXCR4 for infection (the R5 virus SHIV-162P3, the X4 virus SHIV-KU1 and the R5X4 virus SHIV-89.6P), and it also protected against SIVmac251 (R5). Protection of half of the test animals was estimated by interpolation to occur at T-1249 concentrations of approximately 40-130 muM, whereas complete protection was observed at 0.1-2 mM. In vitro, T-1249 had substantial breadth of activity against HIV-1 strains from multiple genetic subtypes and in a coreceptor-independent manner. Thus, at 1 muM in a peripheral blood mononuclear cell-based replication assay, T-1249 inhibited all 29 R5 viruses, all 12 X4 viruses and all 7 R5X4 viruses in the test panel, irrespective of their genetic subtype. Combining lower concentrations of T-1249 with other entry inhibitors (CMPD-167, BMS-C, or AMD3465) increased the proportion of test viruses that could be blocked. In the PhenoSense assay, T-1249 was active against 636 different HIV-1 Env-pseudotyped viruses of varying tropism and derived from clinical samples, with IC(50) values typically clustered in a 10-fold range approximately 10 nM. Overall, these results support the concept of using T-1249 as a component of an entry inhibitor-based combination microbicide to prevent the sexual transmission of diverse HIV-1 variants.

In vivo gp41 antibodies targeting the 2F5 monoclonal antibody epitope mediate human immunodeficiency virus type 1 neutralization breadth.

The broadly neutralizing human monoclonal antibodies (MAbs) 2F5 and 4E10, both targeting the highly conserved human immunodeficiency virus type 1 (HIV-1) envelope membrane proximal external region (MPER), are among the MAbs with the broadest heterologous neutralizing activity and are of considerable interest for HIV-1 vaccine development. We have identified serum antibodies from an HIV-infected subject that both were broadly neutralizing and specifically targeted MPER epitopes that overlap the 2F5 epitope. These MPER-specific antibodies were made 15 to 20 months following transmission and concomitantly with the development of autoantibodies. Our findings suggest that multiple events (i.e., genetic predisposition and HIV-1 immune dysregulation) may be required for induction of broadly reactive gp41 MPER antibodies in natural infection.

Initial B-cell responses to transmitted human immunodeficiency virus type 1: virion-binding immunoglobulin M (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia.

A window of opportunity for immune responses to extinguish human immunodeficiency virus type 1 (HIV-1) exists from the moment of transmission through establishment of the latent pool of HIV-1-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus), but, to date, this period has been logistically difficult to analyze. To probe B-cell responses immediately following HIV-1 transmission, we have determined envelope-specific antibody responses to autologous and consensus Envs in plasma donors from the United States for whom frequent plasma samples were available at time points immediately before, during, and after HIV-1 plasma viral load (VL) ramp-up in acute infection, and we have modeled the antibody effect on the kinetics of plasma viremia. The first detectable B-cell response was in the form of immune complexes 8 days after plasma virus detection, whereas the first free plasma anti-HIV-1 antibody was to gp41 and appeared 13 days after the appearance of plasma virus. In contrast, envelope gp120-specific antibodies were delayed an additional 14 days. Mathematical modeling of the earliest viral dynamics was performed to determine the impact of antibody on HIV replication in vivo as assessed by plasma VL. Including the initial anti-gp41 immunoglobulin G (IgG), IgM, or both responses in the model did not significantly impact the early dynamics of plasma VL. These results demonstrate that the first IgM and IgG antibodies induced by transmitted HIV-1 are capable of binding virions but have little impact on acute-phase viremia at the timing and magnitude that they occur in natural infection.

Impact of the enfuvirtide resistance mutation N43D and the associated baseline polymorphism E137K on peptide sensitivity and six-helix bundle structure.

Enfuvirtide (ENF), the first human immunodeficiency virus type 1 (HIV-1) fusion inhibitor approved for clinical use, acts by binding to gp41 heptad repeat 1 (HR1) and preventing its interaction with the viral HR2 region. Treatment-emergent resistance to ENF has been mapped to residues within HR1, and these mutations decrease its susceptibility to ENF and may reduce viral fitness and pathogenesis, although the mechanism for these effects is not clear. N43D, a common ENF resistance mutation, was found in in vitro assays to cause a 5-50-fold in antiviral activity. We introduced this mutation into peptide models and determined the impact of this mutation by circular dichroism and X-ray crystallography. We find that the mutation results in a decrease in the thermal stability of the six-helix bundle and causes a significant change in the HR1-HR2 interface, including a loss of HR2 helicity. These data form a mechanistic basis for the decrease in ENF sensitivity and six-helix bundle stability. The E137K polymorphism, generally present at baseline in patients who develop N43D, partially compensates for the loss of stability, and we show that these residues likely form an ion pair. These data form a framework for understanding the impact of resistance mutations on viral fitness and pathogenesis and provide a pathway for the development of novel fusion inhibitor peptides.

Safety and efficacy of enfuvirtide in combination with darunavir-ritonavir and an optimized background regimen in treatment-experienced human immunodeficiency virus-infected patients: the below the level of quantification study.

Enfuvirtide is the first fusion and entry inhibitor approved for use for the treatment of human immunodeficiency virus (HIV) type 1 infection and as such represents a novel class of agents. For the population of patients experienced with three antiretroviral classes, enfuvirtide provides an additional option for treatment. This prospective, open-label, 24-week, single-arm trial assessed the efficacy and safety of enfuvirtide (90 mg injected subcutaneously twice daily) in combination with darunavir-ritonavir (600/100 mg administered orally twice daily) in triple-antiretroviral-class-experienced adults failing their current regimen. The primary efficacy endpoint was the proportion of participants with plasma HIV RNA loads of <50 copies/ml. Other virological and immunological measures were also evaluated, as were the effects of the baseline viral coreceptor tropism and darunavir phenotype sensitivity scores on the outcomes. At week 24, 60.3%, 72.5%, and 84.0% of 131 participants achieved viral loads of <50 copies/ml and <400 copies/ml and a change from the baseline load of > or =1 log(10) copies/ml, respectively. A baseline viral load of < or =5 log(10) copies/ml was a significant predictor of achieving a viral load of <50 copies/ml at 24 weeks; however, neither background genotype sensitivity nor darunavir phenotype sensitivity was a significant predictor of the achievement of viral loads of <50 copies/ml. Although these findings are limited by the relatively small numbers of participants with darunavir susceptibility changes of > or =10-fold, they suggest that combining enfuvirtide and darunavir-ritonavir with an optimized background regimen in triple-class experienced participants naïve to these agents can result in positive virological and immunological responses regardless of most baseline parameters.

Characterization of determinants of genotypic and phenotypic resistance to enfuvirtide in baseline and on-treatment HIV-1 isolates.

BACKGROUND: Enfuvirtide (ENF) is the first of a novel class of drugs that block HIV gp41-mediated viral fusion to host cells. Viruses with mutations at positions 36-38 in HIV-1 gp41 and/or reduced susceptibility to ENF have been selected both in vitro and in vivo. METHODS: An analysis of baseline and on-treatment ENF susceptibility in virus samples from Phase II clinical trial patients treated with ENF as functional monotherapy for 28 days (TRI-003) or in combination with oral antiretrovirals for >/= 48 weeks (T20-205, T20-206 and T20-208). Population sequencing identified amino acid (aa) substitutions at positions 36-45 of gp41 in plasma HIV-1. ENF susceptibility of virus isolates was tested in the cMAGI assay and viral DNA was sequenced for selected isolates. RESULTS: HIV-1 gp41 aa 36-45 were highly conserved in virus from ENF-naive patients, except for a 15% incidence of N42S which did not reduce sensitivity to ENF. Virus from patients experiencing viral load rebound exhibited reduced susceptibility to ENF and substitutions in gp41 aa 36-45. The most common substitutions observed on treatment were at positions 36, 38, 40, 42 and 43. On-treatment changes in the phenotypic susceptibility of virus isolates to ENF were generally associated with genotypic changes in aa 36-45. There was a relatively lower incidence of ENF resistance in patients with baseline sensitivity to more oral antiretrovirals in comparison to patients sensitive to fewer antiretrovirals. CONCLUSIONS: These data identify the importance of HIV-1 gp41 aa 36-45 in the emergence of resistance to ENF.

Initiation of human immunodeficiency virus type 1 (HIV-1) transcription is inhibited by noncytolytic CD8 suppression.

The replication of human immunodeficiency virus type 1 (HIV-1) can be inhibited by noncytolytic CD8(+) T cell mediated suppression, an immune response that specifically targets HIV-1 gene expression. Clinical studies demonstrate that this immune response may play an important role in the host defense against HIV infection. In this study, we examined the distinct steps in viral gene expression for inhibition by noncytolytic CD8(+) T cells. A primary HIV-1 infection system of CD4(+) enriched peripheral blood mononuclear cells was utilized to examine the HIV-1 life cycle as a relevant ex vivo system. Established CD8(+) T cell lines from two HIV(+) long-term nonprogressors were used to examine differences at the level of transcriptional initiation and elongation of the HIV genome. This infection system coupled with the results from real-time measurement of newly transcribed RNA transcripts determined that there was a significant decrease (5-8 fold) in short intracellular viral RNA transcripts. These data strongly favor a role for the initiation of virus transcription in noncytolytic CD8(+) T cell mediated suppression.

Association between specific enfuvirtide resistance mutations and CD4 cell response during enfuvirtide-based therapy.

Analysis of CD4 cell responses during 48 weeks of enfuvirtide therapy after virological failure (analysis of covariance) demonstrated significant associations between V38 mutations (n = 58 subjects) and continued CD4 cell increases and between Q40 mutations (n = 8) and loss of CD4 cell benefit (+34 versus -95 cells/mul, P < 0.001). Subjects with N43 (n = 20) or other mutations (n = 48) had intermediate CD4 cell responses. These data suggest that key enfuvirtide resistance mutations may be associated with reduced viral pathogenicity in vivo.

Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus.

Enfuvirtide (ENF), the first approved fusion inhibitor (FI) for HIV, is a 36-aa peptide that acts by binding to the heptad repeat 1 (HR1) region of gp41 and preventing the interaction of the HR1 and HR2 domains, which is required for virus-cell fusion. Treatment-acquired resistance to ENF highlights the need to create FI therapeutics with activity against ENF-resistant viruses and improved durability. Using rational design, we have made a series of oligomeric HR2 peptides with increased helical structure and with exceptionally high HR1/HR2 bundle stability. The engineered peptides are found to be as much as 3,600-fold more active than ENF against viruses that are resistant to the HR2 peptides ENF, T-1249, or T-651. Passaging experiments using one of these peptides could not generate virus with decreased sensitivity, even after >70 days in culture, suggesting superior durability as compared with ENF. In addition, the pharmacokinetic properties of the engineered peptides were improved up to 100-fold. The potent antiviral activity against resistant viruses, the difficulty in generating resistant virus, and the extended half-life in vivo make this class of fusion inhibitor peptide attractive for further development.

HIV-1 coreceptor use in triple-class treatment-experienced patients: baseline prevalence, correlates, and relationship to enfuvirtide response.

OBJECTIVE: We wished to assess, in heavily treatment-experienced patients, the prevalence of and baseline characteristics associated with HIV-1 coreceptor use and their relationship to responses to enfuvirtide treatment. METHODS: Samples were obtained from participants in phase 3 studies of enfuvirtide. Multiple logistic regression and analysis of covariance were performed on data for baseline coreceptor use, virological and immunological response, and changes in coreceptor use during treatment. RESULTS: Baseline envelopes were phenotyped for 724 patients; 50% harbored R5 strains, 48% harbored dual/mixed (D/M) strains, and 2% harbored X4 strains. D/M strains were associated with significantly lower CD4(+) cell counts but comparable viral loads, compared with R5 strains (P=.0005). Virological and immunological responses to enfuvirtide-based treatment showed no correlation with baseline coreceptor use. Changes in virus tropism from D/M to R5 strains during treatment were common, particularly in patients who received enfuvirtide (27%, vs. 14% who received no enfuvirtide; P<.05). CONCLUSION: At baseline, D/M strains were associated with lower CD4(+) cell counts but similar viral loads, compared with R5 strains, and were common across CD4(+) cell count strata. The comparable virological and immunological responses and bias toward shifts from D/M to R5 strains in patients who received enfuvirtide support its use in triple-class treatment-experienced patients and its study as a therapeutic partner for coreceptor-binding inhibitors.

T-1249 retains potent antiretroviral activity in patients who had experienced virological failure while on an enfuvirtide-containing treatment regimen.

BACKGROUND: T-1249 is a 39-amino acid synthetic peptide fusion inhibitor (FI) shown to preserve antiretroviral activity in vitro against human immunodeficiency virus (HIV) isolates that have decreased susceptibility to enfuvirtide (ENF). METHODS: A 10-day phase 1/2 study of the safety and antiretroviral activity of T-1249 was conducted in 53 HIV-1-infected adults with detectable viremia while on an ENF-containing treatment regimen. RESULTS: From FI-naive baseline levels, the geometric mean (GM) decrease in susceptibility to ENF was 116.3-fold, and the GM decrease in susceptibility to T-1249 was 2.0-fold. Patients continued to administer their failing treatment regimen but replaced ENF with T-1249 at a dose of 192 mg/day. T-1249 was generally well tolerated; injection site reactions, which were generally mild, were the most commonly reported adverse event (64% of patients). The median change from levels of HIV-1 RNA at baseline to levels on day 11 was -1.26 log(10) copies/mL (95% confidence interval, -1.40 to -1.09 log(10) copies/mL); on day 11, a decrease from baseline HIV-1 RNA levels of >/=1.0 log(10) copies/mL was seen in 73% of patients. Antiretroviral activity, as measured by levels of HIV-1 RNA, was not predicted by baseline susceptibility to T-1249 or to ENF; genotypic substitutions that emerged during T-1249 treatment were identified in virus from some patients. CONCLUSIONS: These results indicate that FIs constitute an expanding class of antiretroviral agents with the potential to be sequenced.

Resistance to enfuvirtide, the first HIV fusion inhibitor.

Fusion inhibitors are a new class of antiretroviral drugs (ARVs) for the treatment of human immunodeficiency virus infection. Enfuvirtide is the first in this class to reach market approval. Fusion inhibitors block the last step in the three-step viral entry process consisting of attachment, co-receptor binding and fusion, thereby preventing viral capsid entry into the host cell. Enfuvirtide has a unique mechanism of action and high viral target specificity, and in clinical trials has been shown to exhibit both high efficacy and low toxicity. Enfuvirtide is a peptide mimetic of an essential region within viral envelope glycoprotein gp41 that functions by blocking gp41 structural rearrangements at a transitional pre-fusion conformation. Although different clinical isolates show variation in susceptibility to enfuvirtide, primary resistance has not been observed, and thus enfuvirtide-naive isolates remain clinically sensitive. Acquired resistance centres round a 10 amino acid motif between residues 36 and 45 in gp41 that forms part of the binding site of enfuvirtide. The 10 amino acid motif is critical for viral fusion, and enfuvirtide-resistant mutants show poor replicative capacity compared with wild type. Reversion to a wild-type, drug-sensitive state has been reported following enfuvirtide withdrawal.

Mechanisms for HIV-1 Entry: Current Strategies to Interfere with This Step.

Striking reductions in HIV replication, in vivo, by potent combinations of antiretroviral therapies (ART) are the most significant contributor to the decline in HIV morbidity and mortality. Unfortunately, HIV is not eradicated and rebounds quickly when therapy is stopped. Drug toxicity and the emergence of resistant virus cause virologic treatment failure in 40% to 60% of patients, underscoring the need for improved therapeutic modalities. Recent advances regarding the mechanisms and molecules involved in HIV entry have stimulated development of novel therapeutics. A phase I/IIB trial of an HIV-1 fusion inhibitor demonstrated potent inhibition of virus replication, providing proof of the concept that HIV entry can be blocked in vivo. The development of entry inhibitors and their addition to the armamentarium of HIV therapeutics will likely lead to more efficacious cocktails of antiretroviral agents for salvage therapy of antiretroviral-experienced patients, as well as for treatment of antiretroviral-naive patients.