Shetha Shukair

Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

Human Immunodeficiency Virus Type 1 Is Trapped by Acidic but Not by Neutralized Human Cervicovaginal Mucus

ABSTRACT To reliably infect a primate model for human immunodeficiency virus (HIV), ∼10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here, we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1,000-fold more slowly than it does in water. Lactobacilli acidify CVM to pH ∼4 by continuously producing lactic acid. At this acidic pH, we found that lactic acid, but not HCl, abolished the negative surface charge on HIV without lysing the virus membrane. In contrast, in CVM neutralized to pH 6 to 7, as occurs when semen temporarily neutralizes the vagina, HIV maintained its native surface charge and diffused only 15-fold more slowly than it would in water. Thus, methods that can maintain both a high lactic acid content and acidity for CVM during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.

Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II

ABSTRACT Alterations in glucose metabolism have been demonstrated for diverse disorders ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. HKI and II can bind to mitochondria through their N-terminal hydrophobic regions, and their overexpression in tissue culture protects against cell death. In order to determine the relative contributions of mitochondrial binding and glucose-phosphorylating activities of HKs to their overall protective effects, we expressed full-length HKI and HKII, their truncated proteins lacking the mitochondrial binding domains, and catalytically inactive proteins in tissue culture. The overexpression of full-length proteins resulted in protection against cell death, decreased levels of reactive oxygen species, and possibly inhibited mitochondrial permeability transition in response to H2O2. However, the truncated and mutant proteins exerted only partial effects. Similar results were obtained with primary neonatal rat cardiomyocytes. The HK proteins also resulted in an increase in the phosphorylation of voltage-dependent anion channel (VDAC) through a protein kinase Cε (PKCε)-dependent pathway. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKCε.

Human cervicovaginal mucus contains an activity that hinders HIV-1 movement

Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.

Inhibition of the Transport of HIV In Vitro Using a pH-Responsive Synthetic Mucin-Like Polymer System

In conjunction with the routine role of delivering the active ingredient, carefully designed drug delivery vehicles can also provide ancillary functions that augment the overall efficacy of the system. Inspired by the ability of the cervicovaginal mucus to impede the movement of HIV virions at acidic pH, we have engineered a pH-responsive synthetic polymer that shows improved barrier properties over the naturally occurring cervicovaginal mucus by inhibiting viral transport at both acidic and neutral pH. The pH-responsive synthetic mucin-like polymer is constructed with phenylboronic acid (PBA) and salicylhydroxamic acid (SHA), each individually copolymerized with a 2-hydroxypropyl methacrylamide (pHPMA) polymer backbone. At pH 4.8, the crosslinked polymers form a transient network with a characteristic relaxation time of 0.9 s and elastic modulus of 11 Pa. On addition of semen, the polymers form a densely crosslinked elastic network with a characteristic relaxation time greater than 60 s and elastic modulus of 1800 Pa. Interactions between the PBA-SHA crosslinked polymers and mucin at acidic pH showed a significant increase in elastic modulus and crosslink lifetime (p < 0.05). A transport assay revealed that migration of HIV and cells was significantly impeded by the polymer network at pH ≥ 4.8 with a diffusion coefficient of 1.60 x 10(-4) μm(2)/s for HIV. Additionally, these crosslinked polymers did not induce symptoms of toxicity or irritation in either human vaginal explants or a mouse model. In summary, the pH-responsive crosslinked polymer system reported here holds promise as a class of microbicide delivery vehicle that could inhibit the transport of virions from semen to the target tissue and, thereby, contribute to the overall activity of the microbicide formulation.

Influence of hormones and HIV infection on viral transport.

Background Inhibiting transport of virions within the female reproductive tract is an attractive mechanism for transmission prevention. The mucosal environment varies with menstrual cycle and concurrent bacterial vaginosis (BV). Previous studies of transport within mucosal samples have focused on cervico-vaginal samples in exclusion. Severe BV corresponds to increased incidence of female-to-male HIV-1 transmission although the mechanism remains unclear.

63 MITOCHONDRIAL BINDING AND GLUCOSE PHOSPHORYLATION ARE BOTH NEEDED FOR THE PROTECTIVE EFFECTS OF HEXOKINASE I AND II.

Alterations in glucose metabolism have been demonstrated in diverse disorders, ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. Overexpression of HKI and HKII in tissue culture protects against oxidant-induced cell death. The protective effects of these enzymes are thought to be due to either an increase in glucose phosphorylation or closure of the mitochondrial permeability transition pore (mPTP) as a result of HK binding to the voltage-dependent anion channel (VDAC) on the mitochondria. VDAC is believed to form part of mPTP, the opening of which causes cellular injury. The relative contribution of HK binding to the mitochondria and the increase in glucose phosphorylation to the overall protective effects of HKs are not clear. Furthermore, there is no clear evidence supporting the hypothesis that HK binding to mitochondria inhibits mPTP. To better understand the mechanism(s) behind the protective effects of HKs, we overexpressed full-length HKI and HKII (FL-HKI and FL-HKII, respectively), their truncated proteins lacking the N-terminal hydrophobic domains (Tr-HKI and Tr-HKII, respectively), and catalytically inactive proteins (Mut-HKI and mut-HKII, respectively) in human embryonic kidney (HEK293) cells. The truncated enzymes cannot bind to mitochondria but can phosphorylate glucose, whereas the catalytically inactive enzymes can bind to the mitochondria but do not phosphorylate glucose. The cells overexpressing these constructs were subjected to oxidant stress followed by measurement of mitochondrial membrane potential and cell death. Overexpression of FL-HKI and FL-HKII resulted in complete protection against oxidant-induced loss of mitochondrial membrane potential and cell death (survival percentage of 96 ± 9 and 95 ± 5 for FL-HKI and FL-HKII, respectively). Although overexpression of the truncated and mutant proteins reduced cell death, the degree of protection was about 40 to 50% less than that of the full-length proteins. Furthermore, FL-HKI and FL-HKII inhibited mitochondrial permeability transition (MPT) in the presence of H2O2, whereas the truncated and mutant forms only caused partial inhibition. Similar results were obtained when these proteins were expressed in primary neonatal rat cardiomyocytes using an adenoviral technique. To understand the mechanism for the protective effects of HKs, we measured VDAC phosphorylation in cells overexpressing these proteins. Overexpression of FL-HKI and FL-HKII resulted in a 5- to 10-fold increase in VDAC phosphorylation. The mechanism for VDAC phosphorylation appears to be through PKC-e as inhibitors of this enzyme led to a reversal of this process. These results suggest that both glucose phosphorylation and inhibition of mPTP contribute to the protective effects of HKI and HKII. Furthermore, overexpression of HKI and HKII leads to VDAC phosphorylation in a PKC-e-dependent pathway. These findings bear implications of HK overexpression and binding to the mitochondria as a potential clinical treatment strategy for various forms of human disease.