Stabilization of HIV-1 envelope in the CD4-bound conformation through specific cross-linking of a CD4 mimetic.

CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.

Activity and safety of synthetic lectins based on benzoboroxole-functionalized polymers for inhibition of HIV entry.

Lectins derived from plant and microbial sources constitute a vital class of entry inhibitors that target the oligomannose residues on the HIV envelope gp120. Despite their potency and specificity, success of lectin-based entry inhibitors may be impeded by high manufacturing costs, formulation and potential mitogenicity. Therefore, there exists a gap in the HIV microbicides pipeline that underscores the need for mass producible, synthetic, broad-spectrum, and biocomptabile inhibitors of HIV entry. Here, we present the development of a polymeric synthetic lectin, based on benzoboroxole (BzB), which exhibits weak affinity (∼25 M(-1)) for nonreducing sugars, similar to those found on the HIV envelope. High molecular weight BzB-functionalized polymers demonstrated antiviral activity that increased with an increase in ligand density and molecular weight of the polymer construct, revealing that polyvalency improves activity. Polymers showed significant increase in activity from 25 to 75 mol % BzB functionalization with EC(50) of 15 µM and 15 nM, respectively. A further increase in mole functionalization to 90% resulted in an increase of the EC(50) (59 ± 5 nM). An increase in molecular weight of the polymer at 50 mol % BzB functionalization showed a gradual but significant increase in antiviral activity, with the highest activity seen with the 382 kDa polymer (EC(50) of 1.1 ± 0.5 nM in CEM cells and 11 ± 3 nM in TZM-bl cells). Supplementing the polymer backbone with 10 mol % sulfonic acid not only increased the aqueous solubility of the polymers by at least 50-fold but also demonstrated a synergistic increase in anti-HIV activity (4.0 ± 1.5 nM in TZM-bl cells), possibly due to electrostatic interactions between the negatively charged polymer backbone and the positively charged V3-loop in the gp120. The benzoboroxole-sulfonic acid copolymers showed no decrease in activity in the presence of a seminal concentration of fructose (p > 0.05). Additionally, the copolymers exhibit minimal, if any, effect on the cellular viability, barrier properties, or cytokine levels in human reconstructed ectocervical tissue after 3 days of repeated exposure and did not show pronounced activity against a variety of other RNA and DNA viruses.

Diminishing biofilm resistance to antimicrobial nanomaterials through electrolyte screening of electrostatic interactions.

The extracellular polymer substances (EPS) generated by biofilms confers resistance to antimicrobial agents through electrostatic and steric interactions that hinder molecular diffusion. This resistance mechanism is particularly evident for antibacterial nanomaterials, which inherently diffuse more slowly compared to small organic antibacterial agents. The aim of this study was to determine if a biofilm's resistance to antibacterial nanomaterial diffusion could be diminished using electrolytes to screen the EPS's electrostatic interactions. Anionic (+) alpha-tocopherol phosphate (α-TP) liposomes were used as the antimicrobial nanomaterials in the study. They self-assembled into 700 nm sized structures with a zeta potential of -20 mV that were capable of killing oral bacteria (S. oralis growth inhibition time of 3.34 ± 0.52 h). In a phosphate (-ve) buffer the -ve α-TP liposomes did not penetrate multispecies oral biofilms, but in a Tris (hydroxymethyl)aminomethane (+ve) buffer they did (depth - 12.4 ± 3.6 µm). The Tris did not modify the surface charge of the α-TP nanomaterials, rather it facilitated the α-TP-biofilm interactions through electrolyte screening (Langmuir modelled surface pressure increase of 2.7 ± 1.8 mN/ m). This data indicated that EPS resistance was mediated through charge repulsion and that this effect could be diminished through the co-administration of cationic electrolytes.

In vitro efficacy of essential oil mouthrinse versus dentifrices.

OBJECTIVES: To compare the antimicrobial efficacy and kill penetration of essential oils (EO) mouthrinse versus stannous fluoride, and triclosan dentifrice slurries on saliva-derived biofilms using confocal laser scanning microscopy (CLSM). METHODS: Saliva-derived biofilms were grown for 48h on hydroxyapatite discs using pooled, homogenized saliva from 8 healthy volunteers as the inoculum. The mean thickness of these biofilms was 84µm (range, 23-241µm). CLSM with viability mapping was used to visualize the antimicrobial kill penetration of each treatment regime within a biofilm. RESULTS: At 30s treatment durations, CLSM imaging revealed greater antimicrobial activity and kill penetration of EO mouthrinse compared to sodium fluoride-, stannous fluoride-, and triclosan-containing dentifrice slurries. Quantification of biovolume revealed that EO mouthrinse treatment at 30s resulted in a greater non-viable biovolume proportion (84.6%±15.0%) than other treatment groups. Increasing the treatment duration of the triclosan dentifrice (to 60 and 120s) resulted in better penetration and an increased reduction of viable cells, comparable to EO mouthrinse treatment at 30s duration. Further, CLSM imaging showed that the combined treatment of a non-antimicrobial dentifrice (45s) with EO mouthrinse (30s) showed superior antimicrobial activity (96.2%±3.7%) compared to the antimicrobial triclosan-containing dentifrice used without a mouthrinse step (26.0%±32.0%). CONCLUSIONS: Within typical exposure times, the EO-containing mouthrinse can penetrate deep into the accumulating plaque biofilm compared to the chemotherapeutic dentifrice slurries, and may provide an efficacious alternative to triclosan, when used as an adjunct with a mechanical oral care regimen. CLINICAL SIGNIFICANCE: Using viability mapping and CLSM, this study demonstrated that EO-containing mouthrinse penetrates and kills microorganisms deeper and more effectively in plaque biofilm in typical exposure times when compared to dentifrice chemotherapeutic agents, providing an efficacious alternative to triclosan or stannous fluoride when used as an adjunct to mechanical oral care.

Biophysical analysis of prototype microbicidal gels.

The objective of this study was to evaluate the distribution and retention (deployment) of four prototype vehicles for delivery of prophylactic microbicides against vaginal HIV transmission. Study gels were created with different molecular compositions, producing different biophysical properties governing vaginal deployment. The study employed three techniques: direct rheological measurement of gel properties, direct observation of gel surface coating erosion, and dissolution by a vaginal fluid simulant, and mathematical modeling of gel squeezing flow processes. Results suggest significant differences in extent of vaginal coating after gel application and in erosion of these gel layers due to contact with ambient vaginal fluid and shearing. The relationships between gel rheological properties, coating flow and erosion of coating were not always anticipated from differences in gel molecular composition.

Erosion of microbicide formulation coating layers: effects of contact and shearing with vaginal fluid or semen.

An effective vaginal microbicide formulation must distribute and maintain an epithelial coating layer. The post-application durability of this coating is significantly affected by the vaginal environment. A new in vitro assay quantified coating layer erosion after contact and shear with simulated vaginal fluid or semen. Coating layer persistence and viscosity of both fluid and gel layers were assessed versus time. Five vaginal formulations were studied. In all gels, there was an overall trend of rapid ( approximately 30 min) and significant viscosity loss. Although there were differences across gels and between simulants, greater erosion occurred after contact with the low-pH vaginal fluid simulant (>50% viscosity decrease), as compared to an alkaline semen simulant. These in vitro results suggest significant differences in vivo of vaginal coating retention by the test gels. This new assay can be diversified to create a spectrum of biologically relevant conditions which collectively simulate the natural history of vaginal formulation residence.

Gravity-induced coating flows of vaginal gel formulations: in vitro experimental analysis.

Efficacy of topical microbicidal drug delivery formulations against HIV depends in part on their coating distributions and retention on vaginal epithelium. This study focused on gravity-induced coating flows of vaginal gels, and effects of formulation composition and surface wettability on coating. We hypothesized that presence of a yield stress, and surface wettability, affect coating. Experiments imaged and analyzed coating flows of gels on inclined model hydrophilic or hydrophobic surfaces. The in vitro wettability conditions bracket those believed to exist on vaginal epithelium in vivo. Six commercial vaginal gels were studied: three polyacrylic acid-based (PAA) and three cellulose-based. Our research group uses these gels in complementary human in vivo studies and other in vitro experimental analyses; this study is a first step in linking the in vivo and in vitro measurements. Coating by PAA gels was different from cellulose-based gels: the former exhibited yield stresses, which prevented initial gel shape from deforming during sliding. Coating flows of cellulose gels depended upon surface wettability. The slipping rates of the PAA gels ranked inversely with fitted yield stress values. The coating flow rates of the cellulose gels (hydrophilic surface) did not correlate with consistency index, but ranked inversely with the shear-thinning index. This study introduces a simple methodology for comparing trial formulations and relating their flows to gel constituents and physical properties. It also suggests differences in coating by current commercial gels.

Elicitation of neutralizing antibodies directed against CD4-induced epitope(s) using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein.

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.

Compartmental transport model of microbicide delivery by an intravaginal ring.

Topical antimicrobials, or microbicides, are being developed to prevent HIV transmission through local, mucosal delivery of antiviral compounds. While hydrogel vehicles deliver the majority of current microbicide products, intravaginal rings (IVRs) are an alternative microbicide modality in preclinical development. IVRs provide a long-term dosing alternative to hydrogel use, and might provide improved user adherence. IVR efficacy requires sustained delivery of antiviral compounds to the entire vaginal compartment. A two-dimensional, compartmental vaginal drug transport model was created to evaluate the delivery of drugs from an intravaginal ring. The model utilized MRI-derived ring geometry and location, experimentally defined ring fluxes and vaginal fluid velocities, and biophysically relevant transport theory. Model outputs indicated the presence of potentially inhibitory concentrations of antiviral compounds along the entire vaginal canal within 24 h following IVR insertion. Distributions of inhibitory concentrations of antiviral compounds were substantially influenced by vaginal fluid flow and production, while showing little change due to changes in diffusion coefficients or ring fluxes. Additionally, model results were predictive of in vivo concentrations obtained in clinical trials. Overall, this analysis initiates a mechanistic computational framework, heretofore missing, to understand and evaluate the potential of IVRs for effective delivery of antiviral compounds.

Semi-solid gels function as physical barriers to human immunodeficiency virus transport in vitro.

Vaginal gels may act as physical barriers to HIV during sexual transmission. However, the extent and significance of this effect are not well understood. During male-to-female sexual transmission of HIV, semen containing infectious HIV is present within the lower female reproductive tract. In cases where a topical gel has previously been applied to the vaginal epithelium, virions must move through gel layers before reaching vulnerable tissue. This additional barrier could affect the functioning of anti-HIV microbicide gels and placebos. To better understand HIV transport in gels, we: (1) quantified diffusion coefficients of HIV virions within semi-solid delivery vehicles; and (2) tested the barrier functioning of thin gel layers in a Transwell system. Two gels used as placebos in microbicides clinical trials, hydroxyethyl cellulose (HEC) and methylcellulose (MC), were found to hinder HIV transport in vitro. The diffusion coefficients for HIV virions in undiluted HEC and MC were 4±2 x 10⁻¹² and 7±1 x 10⁻¹² cm²/s, respectively. These are almost 10,000 times lower than the diffusion coefficient for HIV in water. Substantial gel dilution (80%:diluent/gel, v/v) was required before diffusion coefficients rose to even two orders of magnitude lower than those in water. In the Transwell system, gel layers of approximately 150-µm thickness reduced HIV transport. There was a log reduction in the amount of HIV that had breached the Transwell membrane after 0-, 4-, and 8-h incubations. The ability of a gel to function as a physical barrier to HIV transport from semen to tissue will also depend on its distribution over the epithelium and effects of dilution by vaginal fluids or semen. Results here can serve as a baseline for future design of products that act as barriers to HIV transmission. The potential barrier function of placebo gels should be considered in the design and interpretation of microbicides clinical trials.

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Dynamics of HIV neutralization by a microbicide formulation layer: biophysical fundamentals and transport theory.

Topical microbicides are an emerging HIV/AIDS prevention modality. Microbicide biofunctionality requires creation of a chemical-physical barrier against HIV transmission. Barrier effectiveness derives from properties of the active compound and its delivery system, but little is known about how these properties translate into microbicide functionality. We developed a mathematical model simulating biologically relevant transport and HIV-neutralization processes occurring when semen-borne virus interacts with a microbicide delivery vehicle coating epithelium. The model enables analysis of how vehicle-related variables, and anti-HIV compound characteristics, affect microbicide performance. Results suggest HIV neutralization is achievable with postcoital coating thicknesses approximately 100 mum. Increased microbicide concentration and potency hasten viral neutralization and diminish penetration of infectious virus through the coating layer. Durable vehicle structures that restrict viral diffusion could provide significant protection. Our findings demonstrate the need to pair potent active ingredients with well-engineered formulation vehicles, and highlight the importance of the dosage form in microbicide effectiveness. Microbicide formulations can function not only as drug delivery vehicles, but also as physical barriers to viral penetration. Total viral neutralization with 100-mum-thin coating layers supports future microbicide use against HIV transmission. This model can be used as a tool to analyze diverse factors that govern microbicide functionality.