Sifuvirtide, a potent HIV fusion inhibitor peptide

Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC₅₀), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC₅₀) were relatively high, rendering it an ideal anti-HIV agent.A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1_IIIB were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF.In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.     

Optimal drug treatment regimens for HIV depend on adherence

Drug therapies aimed at suppressing the human immunodeficiency virus (HIV) are highly effective, often reducing the viral load to below the limits of detection for years. Adherence to such antiviral regimens, however, is typically far from ideal. We have previously developed a model that predicts optimal treatment regimens by weighing drug toxicity against CD4+ T-cell counts, including the probability that drug resistance will emerge. We use this model to investigate the influence of adherence on therapy benefit. For a drug with a given half-life, we compare the effects of varying the dose amount and dose interval for different rates of adherence, and compute the optimal dose regimen for adherence between 65% and 95%. Our results suggest that for optimal treatment benefit, drug regimens should be adjusted for poor adherence, usually by increasing the dose amount and leaving the dose interval fixed. We also find that the benefit of therapy can be surprisingly robust to poor adherence, as long as the dose interval and dose amount are chosen accordingly.     

Section: bioprocessing and biological engineering gene fragment polymerization for increased yield of recombinant HIV fusion inhibitor enfuvirtide

Fuzeon (Enfuvirtide, T20) is the first fusion inhibitor approved by the FDA of the USA for the treatment of HIV/AIDS in combination with other anti-retroviral drugs. Enfuvirtide is a synthetic peptide that blocks the entry of HIV into healthy host CD₄ cells, which requires very high (90 mg twice daily) therapeutic doses. To increase the yield of Enfuvirtide, a gene polymerization strategy was introduced and recombinant T20 (rT20) was expressed in Escherichia coli as a five copy repeat polypeptide with a histidine-tag. The five copy rT20 was purified by Ni-affinity chromatography and cleaved to single rT20 units by cyanogen bromide. Finally, single rT20 units were purified by reversed phase chromatography giving a yield (400 mg/l) with a purity >95 %, which exhibited specific biological activity similar to Fuzeon.     

Modelling the effects of pre-exposure and post-exposure vaccines in tuberculosis control

Epidemic control strategies alter the spread of the disease in the host population. In this paper, we describe and discuss mathematical models that can be used to explore the potential of pre-exposure and post-exposure vaccines currently under development in the control of tuberculosis. A model with bacille Calmette-Guerin (BCG) vaccination for the susceptibles and treatment for the infectives is first presented. The epidemic thresholds known as the basic reproduction numbers and equilibria for the models are determined and stabilities are investigated. The reproduction numbers for the models are compared to assess the impact of the vaccines currently under development. The centre manifold theory is used to show the existence of backward bifurcation when the associated reproduction number is less than unity and that the unique endemic equilibrium is locally asymptotically stable when the associated reproduction number is greater than unity. From the study we conclude that the pre-exposure vaccine currently under development coupled with chemoprophylaxis for the latently infected and treatment of infectives is more effective when compared to the post-exposure vaccine currently under development for the latently infected coupled with treatment of the infectives.     

In vitro selection and characterization of HIV-1 variants with increased resistance to sifuvirtide, a novel HIV-1 fusion inhibitor

Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide.     

Role of sequence and structure of the hendra fusion protein fusion Peptide in membrane fusion

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion.     

The influenza haemagglutinin-induced fusion cascade: effects of target membrane permeability changes

To define the stages in influenza haemagglutinin (HA)-mediated fusion the kinetics of fusion between cell pairs consisting of single influenza HA-expressing cells and single erythrocytes (RBC) which had been labelled with both a fluorescent lipid (DiI) in the membrane and a fluorescent solute (calcein) in the aqueous space have been monitored. It is shown that release of solute from the target cell occurs, following the formation of the hemi-fusion diaphragm. These results are discussed in terms of a model in which fusion peptide insertion into the target membrane induces lipid stalks, which results in the formation of a hemifusion diaphragm and a fusion pore. Bilayer expansion due to overproduction of these stalks can give rise to collateral damage of target membranes.     

Cholesterol alters the inhibitory efficiency of peptide-based membrane fusion inhibitor

The membrane composition modulates membrane fusion by altering membrane physical properties and the structure, organization and dynamics of fusion proteins and peptides. The journey of developing peptide-based viral fusion inhibitors is often stalled by the change in lipid composition of viral and target membranes. This makes it important to study the role of membrane composition on the organization, dynamics and fusion inhibiting abilities of the peptide-based fusion inhibitors. Cholesterol, an important constituent of mammalian cell membrane, modulates bilayer properties in multiple ways and impart its effect on the membrane fusion. We have previously shown that TG-23 peptide derived from phagosomal coat protein, coronin 1, shows significant inhibition of fusion between membranes without cholesterol. In this work, we have studied the effect of the TG-23 peptide on the polyethylene glycol-mediated membrane fusion in presence of different concentrations of membrane cholesterol. Our results show that the inhibitory effect of TG-23 is being completely reversed in cholesterol containing membranes. We have evaluated the structure, organization, dynamics and depth of penetration of TG-23 in membranes having different lipid compositions and its effect on membrane properties. Our results demonstrate that cholesterol does not affect the secondary structure of the peptide, however, alters the depth of penetration of the peptide and modifies peptide organization and dynamics. The cholesterol dependent change in organization and dynamics of the peptide influences its efficacy in membrane fusion. Therefore, we envisage that the study of peptide organization and dynamics is extremely important to determine the effect of peptide on the membrane fusion.     

Current laboratory assays and in vitro intracellular Th1 and Th2 cytokine synthesis in monitoring antiretroviral therapy of pediatric HIV infection

In 42 HIV-infected children, 8-10 years old, belonging to the category A state of infection, combined antiretroviral therapy (cART) was applied, consisting of AZT, ddC and Saquinavir. At 6 and 12 weeks following the start of cART, the efficacy of treatment was assessed, both by means of current parameters (blood CD4+ cell levels, virus load) and by measuring the intracellular synthesis of some Th1 (interleukin (IL)-2, interferon-gamma) and Th2 (IL-4, IL-10) cytokines, in CD4+ lymphocytes, respectively. Before cART, low values of blood CD4+ cell counts and a mean of about 8000 virus RNA copies ml(-1) of serum were detected, and in addition decreased levels of production of both intracellular Th1 cytokines, associated with increased levels of one of the Th2 cytokines (IL-10), but not of the other (IL-4), were noticed. After cART, earlier improvement of intracellular IL-2, interferon-gamma and IL-10 synthesis in CD4+ cells occurred compared to CD4+ counts and virus load. The usefulness of scoring the rates at which CD4+ lymphocytes are able to synthesize intracellular Th1 or Th2 cytokines, as an additional immune parameter during combined antiretroviral therapy monitoring in pediatric AIDS, is discussed.     

Modelling the relationship between antibody-dependent enhancement and immunological distance with application to dengue

When antibodies raised in response to a particular pathogen bind with immunologically similar pathogens it may facilitate infection through a phenomenon known as antibody-dependent enhancement (ADE). This process occurs between the four serotypes of dengue virus and, furthermore, secondary infection is a major risk factor in dengue hemorrhagic fever (DHF). Theory has suggested that ADE may be responsible for the large immunological distance between dengue serotypes. We investigate this hypothesis using an epidemic model for dengue in which immunological distance and the strength of immune cross-reaction are expressed separately. Cross-enhancement is considered in three alternative forms acting on susceptibility, transmission and mortality. Previous models have shown that transmission and mortality enhancement can lead to periodicity or chaos. We confirm this result for reasonable levels of susceptibility and transmission enhancement but not for mortality enhancement. We also show that when the two strains have identical basic reproductive numbers no form of enhancement leads to competitive exclusion. When the two strains have different basic reproductive numbers susceptibility or transmission enhancement allow strains with greater immunological similarity to stably coexist but mortality enhancement forces strains to be more distinct. All three forms of enhancement can be associated with DHF and we conclude that mortality enhancement must be dominant if ADE really is responsible for the immunological distance between dengue serotypes.     

Role of neuraminidase-dependent adherence in Bacteroides fragilis attachment to human epithelial cells

Of 50 B. fragilis strains isolated from clinical samples we have demonstrated that 24 (48%) possess an adhesin that mediates a neuraminidase-dependent attachment of B. fragilis to mammalian epithelial cells, but does not mediate any association with human polymorphonuclear leucocytes. This ligand interacts with a mammalian cell receptor that contains a galactoside residue, exposed after neuraminidase pretreatment. Our results suggest a possible role for cell associated neuraminidase in mediating a two step adherence mechanism.     

Broad antiviral activity and crystal structure of HIV-1 fusion inhibitor sifuvirtide

Sifuvirtide (SFT) is an electrostatically constrained α-helical peptide fusion inhibitor showing potent anti-HIV activity, good safety, and pharmacokinetic profiles, and it is currently under phase II clinical trials in China. In this study, we demonstrate its potent and broad anti-HIV activity by using diverse HIV-1 subtypes and variants, including subtypes A, B, and C that dominate the AIDS epidemic worldwide, and subtypes B', CRF07_BC, and CRF01_AE recombinants that are currently circulating in China, and those possessing cross-resistance to the first and second generation fusion inhibitors. To elucidate its mechanism of action, we determined the crystal structure of SFT in complex with its target N-terminal heptad repeat region (NHR) peptide (N36), which fully supports our rational inhibitor design and reveals its key motifs and residues responsible for the stability and anti-HIV activity. As anticipated, SFT adopts fully helical conformation stabilized by the multiple engineered salt bridges. The designing of SFT also provide novel inter-helical salt bridges and hydrogen bonds that improve the affinity of SFT to NHR trimer. The extra serine residue and acetyl group stabilize α-helicity of the N-terminal portion of SFT, whereas Thr-119 serves to stabilize the hydrophobic NHR pocket. In addition, our structure demonstrates that the residues critical for drug resistance, located at positions 37, 38, 41, and 43 of NHR, are irreplaceable for maintaining the stable fusogenic six-helix bundle structure. Our data present important information for developing SFT for clinical use and for designing novel HIV fusion inhibitors.