Elizabeth A. Henning

A Maraviroc-Resistant HIV-1 with Narrow Cross-Resistance to Other CCR5 Antagonists Depends on both N-Terminal and Extracellular Loop Domains of Drug-Bound CCR5

ABSTRACT CCR5 antagonists inhibit HIV entry by binding to a coreceptor and inducing changes in the extracellular loops (ECLs) of CCR5. In this study, we analyzed viruses from 11 treatment-experienced patients who experienced virologic failure on treatment regimens containing the CCR5 antagonist maraviroc (MVC). Viruses from one patient developed high-level resistance to MVC during the course of treatment. Although resistance to one CCR5 antagonist is often associated with broad cross-resistance to other agents, these viruses remained sensitive to most other CCR5 antagonists, including vicriviroc and aplaviroc. MVC resistance was dependent upon mutations within the V3 loop of the viral envelope (Env) protein and was modulated by additional mutations in the V4 loop. Deep sequencing of pretreatment plasma viral RNA indicated that resistance appears to have occurred by evolution of drug-bound CCR5 use, despite the presence of viral sequences predictive of CXCR4 use. Envs obtained from this patient before and during MVC treatment were able to infect cells expressing very low CCR5 levels, indicating highly efficient use of a coreceptor. In contrast to previous reports in which CCR5 antagonist-resistant viruses interact predominantly with the N terminus of CCR5, these MVC-resistant Envs were also dependent upon the drug-modified ECLs of CCR5 for entry. Our results suggest a model of CCR5 cross-resistance whereby viruses that predominantly utilize the N terminus are broadly cross-resistant to multiple CCR5 antagonists, whereas viruses that require both the N terminus and antagonist-specific ECL changes demonstrate a narrow cross-resistance profile.

Phenotypic and Immunologic Comparison of Clade B Transmitted/Founder and Chronic HIV-1 Envelope Glycoproteins

ABSTRACT Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4+ T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.

Primary Infection by a Human Immunodeficiency Virus with Atypical Coreceptor Tropism

ABSTRACT The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5 Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.

Engineering meniscus structure and function via multi-layered mesenchymal stem cell-seeded nanofibrous scaffolds

Despite advances in tissue engineering for the knee meniscus, it remains a challenge to match the complex macroscopic and microscopic structural features of native tissue, including the circumferentially and radially aligned collagen bundles essential for mechanical function. To mimic this structural hierarchy, this study developed multi-lamellar mesenchymal stem cell (MSC)-seeded nanofibrous constructs. Bovine MSCs were seeded onto nanofibrous scaffolds comprised of poly(ε-caprolactone) with fibers aligned in a single direction (0° or 90° to the scaffold long axis) or circumferentially aligned (C). Multi-layer groups (0°/0°/0°, 90°/90°/90°, 0°/90°/0°, 90°/0°/90°, and C/C/C) were created and cultured for a total of 6 weeks under conditions favoring fibrocartilaginous tissue formation. Tensile testing showed that 0° and C single layer constructs had stiffness values several fold higher than 90° constructs. For multi-layer groups, the stiffness of 0°/0°/0° constructs was higher than all other groups, while 90°/90°/90° constructs had the lowest values. Data for collagen content showed a general positive interactive effect for multi-layers relative to single layer constructs, while a positive interaction for stiffness was found only for the C/C/C group. Collagen content and cell infiltration occurred independent of scaffold alignment, and newly formed collagenous matrix followed the scaffold fiber direction. Structural hierarchies within multi-lamellar constructs dictated biomechanical properties, and only the C/C/C constructs with non-orthogonal alignment within layers featured positive mechanical reinforcement as a consequence of the layered construction. These multi-layer constructs may serve as functional substitutes for the meniscus as well as test beds to understand the complex mechanical principles that enable meniscus function.

Effects of Mesenchymal Stem Cell and Growth Factor Delivery on Cartilage Repair in a Mini-Pig Model

Objective We have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor–β3 [TGF-β3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone. Design Trochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-β3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. Results Treatment with TGF-β3 led to a marked increase in positive staining for collagen type II within defects (P < 0.05), while delivery of MSCs did not (P > 0.05). Neither condition had an impact on other histological semiquantitative scores (P > 0.05), and inclusion of MSCs led to significantly less defect fill (P < 0.05). For all measurements, no synergistic interaction was found between TGF-β3 and MSC treatment when they were delivered together (P > 0.05). Conclusions At this early healing time point, treatment with TGF-β3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-β3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

Organized nanofibrous scaffolds to mimic the macroscopic curvature of the meniscus

The menisci are crescent-shaped fibrocartilaginous tissues featuring highly aligned collagen arranged in a circumferential fashion to allow transmission of loads across the knee joint. To mimic this macroscopic orientation, we developed a novel electrospinning method to collect circumferentially aligned (CircAl) fibers, quantified their structure and mechanics, and compared them to linearly aligned (LinAl) scaffolds. Fiber orientation of CircAl scaffolds varied considerably as a function of position (p<;0.05). Conversely, LinAl fibers did not vary over a similar range (p>;0.05). Alterations in cellular alignment on a macroscopic scale were observed on the CircAl scaffolds. Mechanical analysis of the CircAl scaffolds revealed significant interactions between scaffold length and region (p<;0.05). No such differences could be detected in LinAl specimens (p>;0.05). In conclusion, we developed electrospun nanofibrous scaffolds with a spatially varying macroscopic fiber orientation, creating a gradient in fiber alignment and differences in tensile properties over a macroscopic scale. These organized nanofibrous scaffolds can potentially direct the formation of an anatomic meniscus construct with structure and function that vary across a large anatomic expanse.

Fabrication of Organized Nanofibrous Scaffolds to Mimic the Macroscopic Curvature of the Meniscus: Structure and Mechanics

The menisci are crescent-shaped fibrocartilaginous tissues which function to transmit and distribute loads between the femur and tibia of the knee joint. As such, the meniscus experiences complex loads, including tension, compression, and shear. Meniscus function in tension arises from an organized microstructure — bundles of highly aligned collagen circumnavigate the tissue between insertion sites on the tibial plateau (1). These aligned collagen bundles endow the tissue with mechanical properties that are highly anisotropic, and highest in the primary collagen orientation (2). Commercial products to replace the meniscus lack this unique structure and organization (3,4). To address engineering the knee meniscus, we have developed aligned nanofibrous scaffolds that can recapitulate this mechanical anisotropy (5,6). However promising, fibers within these scaffolds are unidirectional, while the fibers within the native tissue have a pronounced c-shaped macroscopic organization. To mimic this macroscopic orientation, we developed a new electrospinning method to collect organized fibers on a planar spinning disc (7). The objective of this study was to quantify the structure and mechanics of nanofibrous scaffolds collected using this novel technique and compare the data to aligned scaffolds obtained from a traditional electrospinning approach. We hypothesized that these circumferentially aligned (CircAl) scaffolds would behave similarly to linearly aligned (LinAl) scaffolds on short length scales, but exhibit marked differences in mechanics as the length scale increased.Copyright