Immunostimulatory effects on THP-1 cells by peptide or protein pharmaceuticals associated with injection site reactions.

Injection site reaction (ISR) is a common side-effect associated with the use of peptide or protein pharmaceuticals. These types of pharmaceuticals-induced activation of antigen-presenting cells is assumed to be a key step in the pathogenesis of immune-mediated ISR. The present study was designed to evaluate the immunostimulatory properties of peptide or protein pharmaceuticals using human monocytic THP-1 cells. Here, THP-1 cells, with or without phorbol-12-myristate-13-acetate (PMA) pretreatment, were exposed to enfuvirtide and glatiramer acetate (positive controls) or evolocumab (negative control) for 6 or 24 h. PMA treatment differentiated non-adherent monocytic THP-1 (nTHP-1) cells into adherent macrophagic THP-1 (pTHP-1) cells that highly express CD11b and CD36. Enfuvirtide increased the release of cytokines, e.g. TNFα, MIP-1ß, and MCP-1, and expression of CD86 and CD54 on nTHP-1 cells at 24 h. Similar immunostimulatory properties of glatiramer acetate were observed both in the nTHP-1 and pTHP-1 cells at 6 h, but the responses were very weak in the pTHP-1 cells. Evolocumab did not affect cytokine secretion or cell surface marker expression in either cell type. Taken together, these in vitro THP-1 cell assays revealed the immunostimulatory properties of enfuvirtide and glatiramer acetate. This assay platform thus could serve as a powerful tool in evaluating potential immune-related ISR risks of peptide or protein pharmaceuticals in humans.

HIV-1 inhibitory properties of eCD4-Igmim2 determined using an Env-mediated membrane fusion assay.

Human Immunodeficiency Virus-1 (HIV-1) entry is dependent on the envelope glycoprotein (Env) that is present on the virion and facilitates fusion between the envelope and the cellular membrane. The protein consists of two subunits, gp120 and gp41, with the former required for binding the CD4 receptor and either the CXCR4 or CCR5 coreceptor, and the latter for mediating fusion. The requirement of fusion for infection has made Env an attractive target for HIV therapy development and led to the FDA approval of enfuvirtide, a fusion inhibitor. Continued development of entry inhibitors is warranted because enfuvirtide resistant HIV-1 strains have emerged. In this study, a novel HIV-1 fusion assay was validated using neutralizing antibodies and then used to investigate the mechanism of action of eCD4-Igmim2, an HIV-1 inhibitor proposed to cooperatively bind the CD4 binding site and the sulfotyrosine-binding pocket of gp120. Greater reduction in fusion levels was observed with eCD4-Igmim2 in the fusion assay than all of the gp120 antibodies evaluated. Lab adapted isolates, HIV-1HXB2 and HIV-1YU2, were sensitive to eCD4-Igmim2 in the fusion assay, while primary isolates, HIV-1BG505 and HIV-1ZM651 were resistant. These results correlated with greater IC50 values for primary isolates compared to the lab adapted isolates observed in a virus neutralization assay. Analysis of gp120 models identified differences in the V1 and V2 domains that are associated with eCD4-Igmim2 sensitivity. This study highlights the use of a fusion assay to identify key areas for improving the potency of eCD4-Igmim2.

Potent suppression of HIV-1 cell attachment by Kudzu root extract.

There is a constant need to improve antiretrovirals against HIV since therapy is limited by cost, side effects and the emergence of drug resistance. Kudzu is a climbing vine from which the root extract (Pueraria lobata), rich in isoflavones and saponins, has long been used in traditional Chinese medicine for a variety of purposes, from weight loss to alcoholism prevention. Here we show that Kudzu root extract significantly inhibits HIV-1 entry into cell lines, primary human CD4+T lymphocytes and macrophages, without cell-associated toxicity. Specifically, Kudzu inhibits the initial attachment of the viral particle to the cell surface, a mechanism that depends on the envelope glycoprotein gp120 but is independent from the HIV-1 cell receptor CD4 and co-receptors CXCR4/CCR5. This activity seems selective to lentiviruses since Kudzu inhibits HIV-2 and simian immunodeficiency virus, but does not interfere with Hepatitis C, Influenza, Zika Brazil and adenovirus infection. Importantly, depending on the dose, Kudzu can act synergistically or additively with the current antiretroviral cocktails against HIV-1 and can block   viruses resistant to the fusion inhibitor Enfuvirtide. Together our results highlight Kudzu's root extract value as a supplement to current antiretroviral therapy against HIV.

A simple, rapid, and sensitive system for the evaluation of anti-viral drugs in rats.

The lack of small animal models for the evaluation of anti-human immunodeficiency virus type 1 (HIV-1) agents hampers drug development. Here, we describe the establishment of a simple and rapid evaluation system in a rat model without animal infection facilities. After intraperitoneal administration of test drugs to rats, antiviral activity in the sera was examined by the MAGI assay. Recently developed inhibitors for HIV-1 entry, two CXCR4 antagonists, TF14016 and FC131, and four fusion inhibitors, T-20, T-20EK, SC29EK, and TRI-1144, were evaluated using HIV-1(IIIB) and HIV-1(BaL) as representative CXCR4- and CCR5-tropic HIV-1 strains, respectively. CXCR4 antagonists were shown to only possess anti-HIV-1(IIIB) activity, whereas fusion inhibitors showed both anti-HIV-1(IIIB) and anti-HIV-1(BaL) activities in rat sera. These results indicate that test drugs were successfully processed into the rat sera and could be detected by the MAGI assay. In this system, TRI-1144 showed the most potent and sustained antiviral activity. Sera from animals not administered drugs showed substantial anti-HIV-1 activity, indicating that relatively high dose or activity of the test drugs might be needed. In conclusion, the novel rat system established here, "phenotypic drug evaluation", may be applicable for the evaluation of various antiviral drugs in vivo.

Efficacy and safety of raltegravir in treatment-experienced HIV-1-infected patients switching from enfuvirtide-based regimens: 48 week results of the randomized EASIER ANRS 138 trial.

OBJECTIVES: To assess the sustainable efficacy and safety of a switch from enfuvirtide to raltegravir in patients with multidrug-resistant HIV infection. METHODS: One hundred and seventy patients with multidrug-resistant HIV infection and suppressed plasma HIV RNA levels < 400 copies/mL under an enfuvirtide-based regimen were randomized to maintain their regimen or to switch to a raltegravir-based regimen (immediate group) in a 48 week prospective, randomized, open-label trial. At week 24, patients in the maintenance arm also switched to raltegravir (deferred group). Baseline genotypic susceptibility scores (GSSs) were calculated using available historical resistance tests. Efficacy was assessed by the cumulative proportion of patients with virological failure, defined as a confirmed plasma HIV RNA ≥ 400 copies/mL up to week 48. The EASIER ANRS 138 trial is registered at ClinicalTrials.gov (NCT00454337). RESULTS: At baseline, 86% of patients had plasma HIV RNA levels <50 copies/mL and 86% had a GSS ≥ 1. Through to week 48, in the on-treatment analysis, only one patient in the immediate group, with a GSS of 0, developed virological failure. At week 48, 90% of patients in both the immediate and deferred groups had plasma HIV-1 RNA levels <50 copies/mL. Median CD4 cell counts remained stable during follow-up. Of note, 12 of 66 (18.2%) patients receiving a regimen combining raltegravir and ritonavir-boosted tipranavir experienced alanine aminotransferase elevations, which led to a switch from tipranavir to darunavir in 8 cases, without discontinuation of raltegravir. CONCLUSIONS: In well-suppressed patients with multidrug-resistant HIV infection, a switch from enfuvirtide to raltegravir is generally well tolerated and has sustained antiviral efficacy when combined with a potent background regimen.

LFA-1 antagonists as agents limiting human immunodeficiency virus type 1 infection and transmission and potentiating the effect of the fusion inhibitor T-20.

Adhesion molecules are known to play major roles in the initiation and stabilization of cell-to-cell contacts during the immunological response. Human immunodeficiency virus type 1 (HIV-1) exploits those interactions to facilitate infection and propagation processes. The primary objective of the present study was to investigate the ability of antagonists specific for lymphocyte function-associated antigen 1 (LFA-1) to diminish HIV-1 infection and transmission. We demonstrate here that LFA-1 antagonists can significantly reduce HIV-1 replication in primary human cells and virus propagation by affecting cell-to-cell interactions. Moreover, the inhibition of LFA-1-mediated adhesion events also potentiates the antiviral efficacy of the peptide fusion inhibitor T-20. Altogether, our data suggest that LFA-1 antagonists represent promising antiviral agents. Antiadhesion therapy could be considered a complementary strategy targeting cellular functions essential for HIV-1 spreading and against which the combined therapy currently used displays a limited efficacy.

HIV type-1 entry inhibitors with a new mode of action.

BACKGROUND: The development of antiviral drugs has provided crucial new means to mitigate or relieve the debilitating effects of many viral pathogens. Regular use of these drugs has led to generation of resistant strains, making the control of many viral infections very difficult, particularly in HIV-seropositive and AIDS patients. A rich source for the discovery of new HIV infection inhibitors has been, and continues to be, the 'mining' of the large diversity of compounds already available in nature, and specifically those from botanical extracts. METHODS: Using a newly developed direct binding assay with mass spectrometry technology (direct analysis in real-time time-of-flight mass spectrometry), we were able to show that compounds present in extracts of elderberry, cinnamon and green tea bind to and block HIV type-1 (HIV-1) infection in target cells. RESULTS: The compounds that blocked HIV-1 infection were flavonoids and A-type proanthocyanidins. The 50% inhibitory concentration values of these extracts ranged from 0.5 to 201 microg/ml for four different HIV-1 serotypes. Interaction matrices with the elderberry extract and enfuvirtide, a peptide HIV-1 fusion inhibitor, revealed significant super additive effects. This indicates that the compounds in elderberry that prevent HIV-1 infection are likely to bind to viral glycoproteins other than gp41 (the binding site for enfuvirtide). CONCLUSIONS: Optimized elderberry, green tea and cinnamon extracts rich in certain flavonoid compounds were shown to block HIV-1 entry and infection in GHOST cells. As such, these types of botanical extracts could provide a starting point for the development of possible safe and reliable cotherapies for HIV-1-positive individuals, as well as for the identification of new small molecules as leading drug candidates for HIV-1 therapeutics and microbicides.

Synergistic efficacy of combination of enfuvirtide and sifuvirtide, the first- and next-generation HIV-fusion inhibitors.

Enfuvirtide and sifuvirtide, the first- and next-generation HIV-fusion inhibitors, contain different functional domains and have distinct target sites. Here, we found that a combination of enfuvirtide and sifuvirtide resulted in potent synergism in inhibiting HIV-1-mediated cell-cell fusion and infection by X4 and R5 as well as enfuvirtide-resistant HIV-1 strains. These findings suggest that application of enfuvirtide and sifuvirtide in combination may improve their efficacy and resistant profile, leading to a reduction of the dosage and frequency of drug use.

Evolution of genotypic and phenotypic resistance during chronic treatment with the fusion inhibitor T-1249.

T-1249 is a peptide HIV fusion inhibitor (FI) previously under development for use in FI-naive and experienced patients. Here we present prospectively planned longitudinal analyses of FI resistance during 48 weeks of T-1249 dosing in patients with extensive prior FI exposure. T1249-105 was a single-arm rollover study in patients with prior resistance to enfuvirtide (ENF) and 10 days of T-1249 functional monotherapy exposure. The phenotype and genotype of plasma virus envelopes were analyzed at baseline and at study weeks 8, 16, and 48. At study entry, viruses had a geometric mean decrease in susceptibility to ENF of 51.8-fold but to T-1249 of 1.8-fold; extensive genotypic resistance to ENF was observed. A median viral load response of - 1.5 log(10) copies/ml was observed at week 2 that was partially sustained (- 0.5 log(10) copies/ml) through 48 weeks. Resistance to T-1249 gradually increased to a geometric mean 92.7-fold decrease from FI-naive baseline; this occurred concomitant with further evolution of gp41 amino acids 36-45, most commonly the G36D (n = 6, 16%) or N43K (n = 9, 24%) substitutions. A novel substitution, A50V (n = 12, 32%), was also common, as were the N126K and S138A substitutions in heptad-repeat 2 (HR-2). These data point toward a primary role for the gp41 36-45 locus in modulating FI binding and suggest that residues in HR-2 may contribute in a more limited manner to development of peptide FI resistance. These data also point toward a substantial genetic barrier and fitness cost to development of resistance to next-generation fusion inhibitors.

Performance of human immunodeficiency virus type 1 gp41 assays for detecting enfuvirtide (T-20) resistance mutations.

The performance of a gp41 assay, created using reagents designed for use with the OpenGene DNA Sequencing System, was evaluated using a panel of plasma samples obtained from enfuvirtide-naive and -experienced human immunodeficiency virus type 1-infected subjects. Resulting sequence data were highly accurate compared to a "home brew" assay and clonal sequence analysis.

A randomized pilot study comparing combination therapy plus enfuvirtide versus a treatment interruption followed by combination therapy plus enfuvirtide.

Most individuals with multidrug-resistant HIV who switch to a new therapeutic regimen containing a single fully effective agent experience incomplete viral suppression. We postulated that interruption of antiretroviral therapy prior to the introduction of such a regimen would improve long-term virological outcomes. Thirty, three-class experienced, enfuvirtide-naive individuals with detectable drug-resistant viraemia were randomized to an immediate enfuvirtide/optimized-background treatment regimen or a 16-week treatment interruption followed by enfuvirtide/optimized-background treatment regimen. The median CD4+ T-cell count and viral load at study entry were 39 cells/mm and 4.72 log10 copies RNA/ml, respectively. There was no evidence of any virological or immunological benefit associated with the interruption. In multivariate analysis, only the baseline phenotypic susceptibility score was predictive of treatment response at week 48 (P=0.009). Only 40% of individuals had evidence of a shift in drug-resistance genotype during the interruption. In summary, interrupting therapy prior to initiating salvage therapy in patients with advanced disease did not result in an improved virological response to enfuvirtide. The collective predictive activity of an enfuvirtide-containing regimen was important in predicting treatment response.

Prospects and strategies for the discovery and development of small-molecule inhibitors of six-helix bundle formation in class 1 viral fusion proteins.

Class I viral fusion proteins have an important role in the fusion of viral membranes with host cell membranes, a critical step in the viral life-cycle. These proteins all have similar structural features and form six-helix bundles in their fusogenic form, a general mechanism of action for virus-cell fusion. The successful discovery of peptide-based inhibitors of fusion proteins, in addition to the US Food and Drug Administration approval of one of these inhibitors as an anti-HIV-1 drug, confirmed that the inhibition of six-helix bundle formation is a viable strategy for identifying antiviral drugs. Because peptide-based drugs have several limitations, research has been undertaken to identify potent small-molecule inhibitors of six-helix bundle formation in a variety of viruses, including HIV-1, human respiratory syncytial virus and measles virus. Small-molecule inhibitors that disrupt six-helix bundle formation and prevent viral infection have been identified. This review will focus on the discovery of these small-molecule inhibitors.

Inhibition of HIV-1 group M and O isolates by fusion inhibitors.

We examined the susceptibility of HIV-1 group M and O isolates to the fusion inhibitors T-20 and T-1249. Unexpectedly, HIV-1 O isolates were as sensitive as group M viruses to inhibition by T-20 but were usually less susceptible to T-1249. Our data suggest that T-20 has broad antiretroviral activity and would be effective in individuals with HIV-1 O infection. However, polymorphisms in gp41 might affect the sensitivity of HIV-1 O to second-generation fusion inhibitors.

Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro.

Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-naïve viruses and resistance to ENF.

Enfuvirtide is active against HIV type 1 group O.

A high diversity within the HR1/HR2 regions of viral gp41 and one natural change (N42D) within the 36-45 aa domain in HIV-1 group O in comparison with HIV-1 group M isolates have led us to suspect that enfuvirtide (ENF) should not be active against HIV-1 group O. We analyzed in vitro and in vivo the antiviral activity of ENF against HIV-1 group O isolates. The IC50 at baseline was 0.15 +/- 0.028 microg/ml in a clinically derived virus specimen. After initiating treatment with ENF, a significant decline in plasma HIV-RNA and CD4 gain was noticed in one patient. Therefore, individuals with HIV-1 group O strains might benefit from ENF therapy.

Immunological and virological study of enfuvirtide-treated HIV-positive patients.

OBJECTIVE: To evaluate the predictive value and evolution of immunological and virological parameters related to HIV entry and pathogenesis in patients receiving enfuvirtide (ENF) plus an optimized regimen. METHODS: A phase III clinical trial substudy of ENF in 22 patients measured virus coreceptor use and sensitivity to ENF, levels of chemokines, cytokines and chemokine receptors, CD38 and HLA-DR expression as markers of T cell activation and ex vivo cell death at baseline and at week 32. RESULTS: Treatment including ENF reduced HIV viral load (P < 0.001) and increased the CD4 cell count in patients that responded (RP) to treatment (n = 14). Significant (P < 0.05) increases were noted in the RP group in CXCR4 and CCR5 expression in CD4 cells without major differences in chemokine and interleukin-7 levels. A decrease in CD38 expression in the absence of HLA-DR changes was observed in CD4 cells. Apoptosis of peripheral blood mononuclear cells was significantly reduced in the RP group. Coreceptor use or ENF sensitivity of virus isolated at baseline was not associated with virus resistance or response to treatment, which appeared to be related to the activation state (HLA-DR expression) of CD4 cells at baseline. CONCLUSION: The outcome of ENF-containing treatment could not be associated with HIV coreceptor use at baseline. CD4 cell activation and viral drug resistance were the only markers of treatment response. Changes induced by ENF-containing regimen were seen in HIV coreceptor expression, including an increase in CCR5+CD4+ cells, a decrease in CD38 T cells and a concomitant reduction of T cell apoptosis.

HIV entry and fusion inhibitors.

Human immunodeficiency virus (HIV) is a retrovirus that is the causative agent of acquired immunodeficiency syndrome (AIDS). Current HIV therapy is based on targeting two critical enzymes in the viral replication machinery: reverse transcriptase and a virally encoded protease. Although mortality rates due to HIV infection have been dramatically reduced, AIDS remains a major health problem throughout the world. The emergence of HIV variants that are resistant to current therapies and potential toxicity associated with their chronic use has highlighted the need for new approaches to HIV inhibition. Identification of the mechanisms underlying viral entry into the host cell has provided a number of novel therapeutic targets and the first of these HIV fusion inhibitors (enfuvirtide [pentafuside, T-20, Fuzeon; Roche Laboratories and Trimeris]) has recently been approved in the US and Europe. This review will focus on recent progress in the development of therapeutics that target the HIV entry process.

Newer treatments for HIV in children.

PURPOSE OF REVIEW: Several new antiretroviral agents have been introduced into pediatric and adult use. This review will summarize information about these new agents and other recent advances in the care of HIV-infected children. RECENT FINDINGS: New drugs like tenofovir, emtricitabine, and enfuvirtide are being rapidly introduced into antiretroviral treatments for adult patients. In addition, some well-established drugs are being modified to make them more convenient (specifically didanosine and stavudine). Unfortunately, pediatric data lag for these new agents, in some cases because of complicated pharmacokinetics in children. At the same time, critical information on how to use established drugs like nelfinavir and efavirenz in younger children is slowly becoming available. Although antiretroviral treatment in children has often been initiated at standard doses of milligrams per kilogram, and susceptibility to drug was presumed in individuals without a previous history of exposure, recent data show that some primary infections are caused by drug-resistant virus, and there is a tremendous variability in serum drug levels in children. Researchers and clinicians should consider the role of baseline antiretroviral susceptibility testing and therapeutic drug monitoring to identify the optimal treatment for each child. SUMMARY: New therapeutic options for children with HIV infection are becoming available as the pharmacokinetics and best strategies for use of newer drugs are studied.

The introduction of the fusion inhibitors in the HAART-regimens.

Interference with HIV entry into target cells provides a novel approach to the treatment of HIV infection. The inhibition of virus fusion with the co-receptor substrate seems the most specific and potentially best way to interfere with HIV infection and replication. The efficacy of the first compound available (enfuvirtide) is evident after the pre-registrative phase II and III studies showing also that the presence of anti gp 41 antibodies in the plasma of the treated patients does not interfere with drug activity. In the failing enfuvirtide treated patients resistant virus was detected in 7/7 after > one year of treatment with genotypic mutations in the HR env domain, however no interclass cross resistance was evidenced. Mutants selected in vivo demonstrated a slight reduction of replication capacity.

Resistance and replicative capacity of HIV-1 strains selected in vivo by long-term enfuvirtide treatment.

Enfuvirtide is the prototype member of a new class of anti HIV-1 agents, the fusion inhibitors (FI). In recent clinical trials, the compound has shown its efficacy in combination with other antiretroviral agents in vivo. However mutant strains resistant to the action of the drug arise quite rapidly in vitro and in vivo. To analyze the process of selection and evolution of HIV-1 strains resistant to enfuvirtide in vivo and to evaluate the impact of resistance on viral fitness, 12 HIV-1 infected subjects treated with T20 (enfuvirtide) for at least one year were included in the study. Gp41-coding sequences were amplified from plasma samples of these subjects at baseline and at different time points during treatment. Seven of the 12 subjects showed selection of gp41 mutations under the selective pressure of enfuvirtide. In particular, these mutations clustered in two distinct regions: (i) a mutational hot-spot localized, as previously described, in the first residues of the N-HR domain, with position number 38 as the most heavily mutated, but including also a G36V, a N42D/T, a N43D, a L44M and a L45M; (ii) other mutations were localized further downstream, within N-HR/C-HR junction and in the C-HR. A recombinant assay specifically designed for the determination of HIV-1 phenotype to FI was developed and validated. Using this assay, we observed that all of the 7 mutated clones displayed substantially reduced susceptibility to T20, IC50 ranging from 0.6 to12.8 microg/ml (>100 fold change). The residues whose mutation was associated with a potent reduction in susceptibility were V38, N42, and N43, other positions such as G36, N44 and L45 playing a minor role. None of the mutant HIV isolates showed cross-resistance to T-1249. By the same method, the HIV-1 replicative capacity of the recombinant clones was tested in the absence of drugs, and for each subject, pre-therapy clones were compared to post-therapy ones. In 3/7 subjects a significant decrease in replicative capacity of the recombinant clones was observed. The phenotypic data from this study suggest that the secondary additional mutations, could be associated with improved resistance or recovery of replicative capacity (compensatory mutations).