Reduced Potency and Incomplete Neutralization of Broadly Neutralizing Antibodies against Cell-to-Cell Transmission of HIV-1 with Transmitted Founder Envs.

Broadly neutralizing antibodies (bNAbs) have been isolated from HIV-1 patients and can potently block infection of a wide spectrum of HIV-1 subtypes. These antibodies define common epitopes shared by many viral isolates. While bNAbs potently antagonize infection with cell-free virus, inhibition of HIV-1 transmission from infected to uninfected CD4+ T cells through virological synapses (VS) has been found to require greater amounts of antibody. In this study, we examined two well-studied molecular clones and two transmitted/founder (T/F) clones for their sensitivities to a panel of bNAbs in cell-free and cell-to-cell infection assays. We observed resistance of cell-to-cell transmission to antibody neutralization that was reflected not only by reductions of antibody potency but also by decreases in maximum neutralization capacity relative to the levels seen with cell-free infections. BNAbs targeting different epitopes exhibited incomplete neutralization against cell-associated virus with T/F Envs, which was not observed with the cell-free form of the same virus. We further identified the membrane-proximal internal tyrosine-based sorting motif as a determinant that can affect the incomplete neutralization of these T/F clones in cell-to-cell infection. These findings indicate that the signal that affects surface expression and/or internalization of Env from the plasma membrane can modulate the presentation of neutralizing epitopes on infected cells. These results highlight that a fraction of virus can escape from high concentrations of antibody through cell-to-cell infection while remaining sensitive to neutralization in cell-free infection. The ability to fully inhibit cell-to-cell transmission may represent an important consideration in the development of antibodies for treatment or prophylaxis.IMPORTANCE In recent years, isolation of new-generation HIV-1 bNAbs has invigorated HIV vaccine research. These bNAbs display remarkable potency and breadth of coverage against cell-free virus; however, they exhibit a diminished ability to block HIV-1 cell-to-cell transmission. The mechanism(s) by which HIV-1 resists neutralization when transmitting through VS remains uncertain. We examined a panel of bNAbs for their ability to neutralize HIV-1 T/F viruses in cell-to-cell infection assays. We found that some antibodies exhibit not only reduced potency but also decreased maximum neutralization capacity or in vitro efficacy against cell-to-cell infection of HIV-1 with T/F Envs compared to cell-free infection of the same virus. We further identified the membrane-proximal internal tyrosine-based sorting motif YXXL as a determinant that can affect the incomplete neutralization phenotype of these T/F clones. When the maximum neutralization capacity falls short of 100%, this can have a major impact on the ability of antibodies to halt viral replication.

Postintegration HIV-1 infection of cervical epithelial cells mediates contact-dependent productive infection of T cells.

The female genital epithelium plays a protective role against invading pathogens; however, sexual transmission of human immunodeficiency virus type 1 (HIV-1) still occurs in healthy women. To model virus-cell interactions in this barrier during sexual transmission, we studied the uptake and infection of ectocervical and endocervical cell lines with cell-free fluorescent protein-expressing recombinant HIV-1 carrying primary transmitted/founder envelope genes. We observed that a subset of both the ectocervical and endocervical epithelial cells become productively infected with cell-free HIV-1 in a CD4-independent manner. In addition, the ability of the semen-derived enhancer of virus infection (SEVI) to enhance virus-epithelial cell interactions was studied. This infection is increased approximately 2-5 fold when inoculation occurs in the presence of SEVI fibrils. Once infected, the epithelial cells are capable of transmitting the virus to target CD4 T cells in coculture in a contact-dependent manner that uses conventional CD4- and coreceptor-dependent entry. The infection of target CD4 T cells only occurs when de novo HIV-1 is produced within the epithelial cells. These findings suggest that a subset of cervical epithelial cells may be actively involved in establishing a systemic HIV infection and should be a target when designing prevention strategies to protect against HIV-1 sexual transmission.

Temporal analysis of hepatitis C virus cell entry with occludin directed blocking antibodies.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization.

Role of institutional climate in fostering diversity in biomedical research workforce: a case study.

This article reviews the barriers to diversity in biomedical research and describes the evolution of efforts to address climate issues to enhance the ability to attract, retain, and develop underrepresented minorities, whose underrepresentation is found both in science and medicine, in the graduate-school biomedical research doctoral programs (PhD and MD/PhD) at Mount Sinai School of Medicine. We also describe the potential beneficial impact of having a climate that supports diversity and inclusion in the biomedical research workforce. The Mount Sinai School of Medicine diversity-climate efforts are discussed as part of a comprehensive plan to increase diversity in all institutional programs: PhD, MD/PhD, and MD, and at the residency, postdoctoral fellow, and faculty levels. Lessons learned from 4 decades of targeted programs and activities at the Mount Sinai School of Medicine may be of value to other institutions interested in improving diversity in the biomedical science and academic medicine workforce.

Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail.

Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T cells through adhesive contacts called virological synapses (VSs). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1-neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry-based infection assays to measure the inhibitory potency of HIV-1-neutralizing monoclonal antibodies (MAb) and HIV-1-neutralizing patient sera against cell-free and VS-mediated infection. To various degrees, anti-Env MAbs exhibited significantly higher 50% inhibitory concentration (IC(50)s) against VS-mediated infection than cell-free infection. Notably, the MAb 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inocula compared to cell-free inocula. A mutant with truncation mutation in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to virus particle maturation but which can still engage in cell-to-cell infection was tested for the ability to resist neutralizing antibodies. The ΔCT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread.

GST pi modulates JNK activity through a direct interaction with JNK substrate, ATF2.

Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75-80% and 25-45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK + ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.

Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts.

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) signaling cascade. They are activated through dual phosphorylation of two residues in the activation loop, a threonine and a tyrosine, by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock, as well as by cytokines and growth factors. Only small amounts of phosphorylated, active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in Escherichia coli, which lack the appropriate upstream kinases. We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active, phosphorylated JNKs suitable for a variety of enzymatic, biophysical and structural characterizations. We utilize N-terminally His-tagged MKK4 that is coexpressed in E. coli with a constitutively active form of MEKK1. This phosphorylated, active His-MKK4 is purified by Ni-NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1α1, JNK1α2 and JNK2α2) that had separately been expressed and purified from E. coli in their inactive forms. These in vitro activated JNKs are phosphorylated on both residues (T183, Y185) in their activation loops and are active towards their substrate, ATF2.