Emily Hanhauser

Nanostructured optical photonic crystal biosensor for HIV viral load measurement

Detecting and quantifying biomarkers and viruses in biological samples have broad applications in early disease diagnosis and treatment monitoring. We have demonstrated a label-free optical sensing mechanism using nanostructured photonic crystals (PC) to capture and quantify intact viruses (HIV-1) from biologically relevant samples. The nanostructured surface of the PC biosensor resonantly reflects a narrow wavelength band during illumination with a broadband light source. Surface-adsorbed biotarget induces a shift in the resonant Peak Wavelength Value (PWV) that is detectable with <10 pm wavelength resolution, enabling detection of both biomolecular layers and small number of viruses that sparsely populate the transducer surface. We have successfully captured and detected HIV-1 in serum and phosphate buffered saline (PBS) samples with viral loads ranging from 104 to 108 copies/mL. The surface density of immobilized biomolecular layers used in the sensor functionalization process, including 3-mercaptopropyltrimethoxysilane (3-MPS), N-gamma-Maleimidobutyryl-oxysuccinimide ester (GMBS), NeutrAvidin, anti-gp120, and bovine serum albumin (BSA) were also quantified by the PC biosensor.

Real-Time Predictions of Reservoir Size and Rebound Time during Antiretroviral Therapy Interruption Trials for HIV

Monitoring the efficacy of novel reservoir-reducing treatments for HIV is challenging. The limited ability to sample and quantify latent infection means that supervised antiretroviral therapy (ART) interruption studies are generally required. Here we introduce a set of mathematical and statistical modeling tools to aid in the design and interpretation of ART-interruption trials. We show how the likely size of the remaining reservoir can be updated in real-time as patients continue off treatment, by combining the output of laboratory assays with insights from models of reservoir dynamics and rebound. We design an optimal schedule for viral load sampling during interruption, whereby the frequency of follow-up can be decreased as patients continue off ART without rebound. While this scheme can minimize costs when the chance of rebound between visits is low, we find that the reservoir will be almost completely reseeded before rebound is detected unless sampling occurs at least every two weeks and the most sensitive viral load assays are used. We use simulated data to predict the clinical trial size needed to estimate treatment effects in the face of highly variable patient outcomes and imperfect reservoir assays. Our findings suggest that large numbers of patients—between 40 and 150—will be necessary to reliably estimate the reservoir-reducing potential of a new therapy and to compare this across interventions. As an example, we apply these methods to the two “Boston patients”, recipients of allogeneic hematopoietic stem cell transplants who experienced large reductions in latent infection and underwent ART-interruption. We argue that the timing of viral rebound was not particularly surprising given the information available before treatment cessation. Additionally, we show how other clinical data can be used to estimate the relative contribution that remaining HIV+ cells in the recipient versus newly infected cells from the donor made to the residual reservoir that eventually caused rebound. Together, these tools will aid HIV researchers in the evaluating new potentially-curative strategies that target the latent reservoir.

Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics

Significance Biosensing technologies have significant impact on medical diagnostics but difficulties in the handling of complex biospecimens, portability, and nonlinearity in dynamic detection range present considerable technical bottlenecks in their translation into clinical settings. Here, we present the nanoplasmonic electrical field-enhanced resonating device (NE2RD) that detects and quantifies multiple biotargets from distinct clinical specimens (i.e., saliva, serum, and whole blood) with a broad linear dynamic range. Unlike conventional platforms, the NE2RD does not require lengthy sample-preparation steps, skilled personnel, or expensive infrastructure. Further, as a model clinical validation study, we monitored chemotherapy effects on viral load for coinfected patients on a single platform. Therefore, the portable NE2RD can be broadly applied to primary care and point-of-care settings with multiple clinical applications. Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients’ homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE2RD), which addresses all these impediments on a single platform. The NE2RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE2RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE2RD’s broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients’ homes.

Printed Flexible Plastic Microchip for Viral Load Measurement through Quantitative Detection of Viruses in Plasma and Saliva

We report a biosensing platform for viral load measurement through electrical sensing of viruses on a flexible plastic microchip with printed electrodes. Point-of-care (POC) viral load measurement is of paramount importance with significant impact on a broad range of applications, including infectious disease diagnostics and treatment monitoring specifically in resource-constrained settings. Here, we present a broadly applicable and inexpensive biosensing technology for accurate quantification of bioagents, including viruses in biological samples, such as plasma and artificial saliva, at clinically relevant concentrations. Our microchip fabrication is simple and mass-producible as we print microelectrodes on flexible plastic substrates using conductive inks. We evaluated the microchip technology by detecting and quantifying multiple Human Immunodeficiency Virus (HIV) subtypes (A, B, C, D, E, G, and panel), Epstein-Barr Virus (EBV), and Kaposi’s Sarcoma-associated Herpes Virus (KSHV) in a fingerprick volume (50 µL) of PBS, plasma, and artificial saliva samples for a broad range of virus concentrations between 102 copies/mL and 107 copies/mL. We have also evaluated the microchip platform with discarded, de-identified HIV-infected patient samples by comparing our microchip viral load measurement results with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) as the gold standard method using Bland-Altman Analysis.

Viremic Control and Viral Coreceptor Usage in Two HIV-1-Infected Persons Homozygous for CCR5 Δ32

Objectives:To determine viral and immune factors involved in transmission and control of HIV-1 infection in persons without functional CCR5. Design:Understanding transmission and control of HIV-1 in persons homozygous for CCR5&Dgr;32 is important given efforts to develop HIV-1 curative therapies aimed at modifying or disrupting CCR5 expression. Methods:We identified two HIV-infected CCR5&Dgr;32/&Dgr;32 individuals among a cohort of patients with spontaneous control of HIV-1 infection without antiretroviral therapy and determined coreceptor usage of the infecting viruses. We assessed genetic evolution of full-length HIV-1 envelope sequences by single-genome analysis from one participant and his sexual partner, and explored HIV-1 immune responses and HIV-1 mutations following virologic escape and disease progression. Results:Both participants experienced viremia of less than 4000 RNA copies/ml with preserved CD4+ T-cell counts off antiretroviral therapy for at least 3.3 and 4.6 years after diagnosis, respectively. One participant had phenotypic evidence of X4 virus, had no known favorable human leukocyte antigen alleles, and appeared to be infected by minority X4 virus from a pool that predominately used CCR5 for entry. The second participant had virus that was unable to use CXCR4 for entry in phenotypic assay but was able to engage alternative viral coreceptors (e.g., CXCR6) in vitro. Conclusion:Our study demonstrates that individuals may be infected by minority X4 viruses from a population that predominately uses CCR5 for entry, and that viruses may bypass traditional HIV-1 coreceptors (CCR5 and CXCR4) completely by engaging alternative coreceptors to establish and propagate HIV-1 infection.

A humanized mouse-based HIV-1 viral outgrowth assay with higher sensitivity than in vitro qVOA in detecting latently infected cells from individuals on ART with undetectable viral loads

Assays that can verify full viral eradication are essential in the context of achieving a cure for HIV/AIDS. In vitro quantitative viral out growth assays (qVOA) are currently the gold standard for measuring latent HIV-1 but these assays often fail to detect very low levels of replication-competent virus. Here we investigated an alternative in vivo approach for sensitive viral detection using humanized mice (hmVOA). Peripheral blood CD4+ T cell samples from HIV subjects on stable ART with undetectable viral loads by RT-PCR were first assayed by in vitro qVOA. Corresponding patient samples in which no virus was detected by qVOA were injected into humanized mice to allow viral outgrowth. Of the five qVOA virus negative samples, four gave positive viral outgrowth in the hmVOA assay suggesting that it is more sensitive in detecting latent HIV-1.

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4 + T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous—increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

Human immunodeficiency virus type 1 persistence following systemic chemotherapy for malignancy

Background Systemic chemotherapies for various malignancies have been shown to significantly, yet transiently, decrease numbers of CD4+ T lymphocytes, a major reservoir for human immunodeficiency virus type 1 (HIV-1) infection. However, little is known about the impact of cytoreductive chemotherapy on HIV-1 reservoir dynamics, persistence, and immune responses. Methods We investigated the changes in peripheral CD4+ T-cell-associated HIV-1 DNA and RNA levels, lymphocyte activation, viral population structure, and virus-specific immune responses in a longitudinal cohort of 15 HIV-1-infected individuals receiving systemic chemotherapy or subsequent autologous stem cell transplantation for treatment of hematological malignancies and solid tumors. Results Despite a transient reduction in CD4+ T cells capable of harboring HIV-1, a 1.7- and 3.3-fold increase in mean CD4+ T-cell-associated HIV-1 RNA and DNA, respectively, were observed months following completion of chemotherapy in individuals on antiretroviral therapy. We also observed changes in CD4+ T-cell population diversity and clonal viral sequence expansion during CD4+ T-cell reconstitution following chemotherapy cessation. Finally, HIV-1 DNA was preferentially, and in some cases exclusively, detected in cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-responsive CD4+ T cells following chemotherapy. Conclusions Expansion of HIV-infected CMV/EBV-specific CD4 + T cells may contribute to maintenance of the HIV DNA reservoir following chemotherapy.

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+T cells expressing CD30

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.

CCR5-Δ32 Heterozygosity, HIV-1 Reservoir Size, and Lymphocyte Activation in Individuals Receiving Long-term Suppressive Antiretroviral Therapy

We conducted a case-controlled study of the associations of CCR5-Δ32 heterozygosity with human immunodeficiency virus type 1 (HIV-1) reservoir size, lymphocyte activation, and CCR5 expression in 114 CCR5(Δ32/WT) and 177 wild-type CCR5 AIDS Clinical Trials Group participants receiving suppressive antiretroviral therapy. Overall, no significant differences were found between groups for any of these parameters. However, higher levels of CCR5 expression correlated with lower amounts of cell-associated HIV-1 RNA. The relationship between CCR5-Δ32 heterozygosity, CCR5 expression, and markers of HIV-1 persistence is likely to be complex and may be influenced by factors such as the duration of ART.