Anthony R. Geonnotti

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.

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.

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.

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.

Measuring macrodiffusion coefficients in microbicide hydrogels via postphotoactivation scanning.

Hydrogels can function as biocompatible, engineered systems to provide controlled release of drugs in vivo. Whether they are employed as gel implants, tissue engineered scaffolds, or delivery systems for transdermal or mucosal delivery, hydrogels often function by controlling the diffusion of particles into or out of their polymer matrices. Biologically relevant predictions of hydrogel functionality in vivo (e.g., pharmacokinetic profiles) require accurate measurements of particle transport. However, determinations of diffusion coefficients in hydrogels are highly influenced by the length scales over which measurements are made. Because of heterogeneity within hydrogel macromolecular structures, local microdiffusion coefficients deviate from ones measured over longer length scales, especially as diffusing particle size increases. Therefore, in vitro measurement of diffusion coefficients should be performed on length scales similar to those of the in vivo transport processes of interest. Several techniques (e.g., FRAP, single particle tracking, scanning microphotolysis, multiple image photography) have been widely utilized to measure particle diffusion within hydrogels, but most provide measurements on molecular or cellular length scales (<30 μm). Other methods (e.g., diffusion chambers, semi-infinite slabs) obtain bulk diffusion measurements over centimeter length scales. While these scales are relevant to many biological processes, they do not capture diffusion on the intermediate range of the order of hundreds of micrometers. This intermediate length scale is prevalent in many biomedical applications and is especially relevant to the HIV/ AIDS prevention field. Here, hydrogels are being used to deliver antiviral microbicidal compounds directly to potential sites of infection as well as to inhibit HIV transport from semen to epithelium. These hydrogels create layers 50–500 μm thick within the lower human female reproductive tract. Understanding the rates at which antiviral agents, as well as HIV itself, migrate through the gel layers is essential to understanding their role in prophylaxis against HIV transmission. This understanding of particle transport within hydrogels can be extended to other drug delivery applications as well. To address this experimental gap, we have created and evaluated a novel method to measure macroscale particle diffusion in hydrogels. Using UV photolysis of caged fluorescent compounds, we generated a region of uniformly high fluorescence intensity with distinct linear margins between regions of very low intensity. The optical creation of a sharp concentration gradient overcomes the limitations of mechanically forming interfaces that are inherent in previous semi-infinite slab configurations and allows diffusion to be studied over intermediate length scales. The sealed chamber and lack of translational movement eliminate convective flow and enable diffusion measurements over long times and, therefore, extended distances. A high-resolution fluorescent scanner quantifies concentration profiles resulting from 1-D diffusion of fluorescent particles outward from the photoactivated linear region. These profiles are imaged over time. A custom MATLAB software program determines a unique solution for the diffusion coefficient by numerically fitting all concentration profiles to the partial differential equation for diffusion. The method has produced accurate, robust quantification of diffusion coefficients over a range of length scales and within a range of liquid or semisolid media. Within the HIV/AIDS prevention field, the method will be applicable to quantify both drug and viral transport within drug delivery vehicles. Moreover, the technique has broad applicability to any processes that involve diffusion through polymeric materials over comparable length scales. This paper presents a description of the method, provides validation measurements, and applies the new technique to measure the diffusion of a small fluorescein molecule (322 Da) and a larger macromolecule (10 kD dextran) in placebo gels used in previous and ongoing microbicide clinical trials.

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.

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-27312/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.