Biology subjects

Wald, A.

Publications and source records attributed to Wald, A..

11 recordsLinked to original sources

bioRxiv2024

Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

Herpes simplex virus (HSV) infection has worldwide public health concerns and lifelong medical impacts. The standard therapy, acyclovir, has limited efficacy in preventing HSV subclinical virus shedding, and drug resistance occurs in immunocompromised patients, highlighting the need for novel therapeutics. HSV manifests in the skin and mucosal epithelium. Here, we found acyclovir significantly less effective in skin-derived keratinocytes than donor-matched fibroblasts. To recapitulate in vivo tissue architecture, we 3D bioprinted human skin equivalents (HSE) in a 96-well plate format amenable for antiviral screening and preclinical testing. We screened a library of 738 compounds with broad targets and mechanisms of action and identified potent antivirals, including 23 known or experimental HSV treatments, validating the translational relevance of our assay. Unlike acyclovir, antivirals against HSV helicase/primase or host replication pathways displayed similar potency across cell types and donor sources in 2D and 3D models. Our 3D bioprinted platform allowed for integrating patient-derived cells and incorporating genetic variability early in drug development. The reduced potency in keratinocytes helps explain the limited benefit acyclovir and its congeners play in reducing sexual transmission. These data indicate that the 3D bioprinted HSE assay platform provides a more physiologically relevant approach to identifying potential antivirals for HSV. One Sentence SummaryHigh-throughput screen using 3D bioprinted human skin equivalents to identify antivirals against HSV and evaluate cell-type specific effects.

bioengineering↗

bioRxiv2024

Treponema pallidum periplasmic and membrane proteins are recognized by circulating and skin CD4+ T cells

BackgroundHistologic and serologic studies suggest the induction of local and systemic Treponema pallidum (Tp)-specific CD4+ T cell responses to Tp infection. We hypothesized that Tp-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. MethodsPBMC collected from 67 participants were screened by IFN{gamma} ELISPOT response to Tp sonicate. Tp-reactive T cell lines from blood and skin were probed for responses to 88 recombinant Tp antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. ResultsWe detected CD4+ T cell responses to Tp sonicate ex vivo. Using Tp-reactive T cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, Tp-specific T cells persisted for at least 6 months in skin and 10 years in blood. ConclusionsTp infection elicits an antigen-specific CD4+ T cell response in blood and skin. Tp-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The Tp antigenic targets we identified may be high priority vaccine candidates.

immunology↗

bioRxiv2024

Cross-species modeling and enhancement of cognitive control with striatal brain stimulation

Brain disorders, particularly mental disorders, might be effectively treated by direct electrical brain stimulation, but clinical progress requires understanding of therapeutic mechanisms. Animal models have not helped, because there are no direct animal models of mental illness. We show a path past this roadblock, by leveraging a common ingredient of most mental disorders: impaired cognitive control. We previously showed that deep brain stimulation (DBS) improves cognitive control in humans. We now reverse translate that result, showing that DBS-like stimulation of the mid-striatum improves cognitive control in rats. Using this model, we identify a mechanism, improvement in domain-general cognitive control, and rule out competing hypotheses such as impulsivity. The rat findings explain prior human results and have immediate implications for clinical practice and future trial design. One Sentence Summary: Developing a reliable animal model of a human brain stimulation therapy reveals that this therapy works by enhancing the brains ability to process conflicting pieces of evidence.

neuroscience↗

bioRxiv2021

Probing Neuro-Endocrine Interactions Through Wireless Magnetothermal Stimulation of Peripheral Organs

Exposure to stress alters hypothalamic-pituitary-adrenal (HPA) axis reactivity; however, it is unclear exactly how or where within the HPA pathway these changes occur. Dissecting these mechanisms requires tools to reliably probe HPA function, particularly the adrenal component, with temporal precision. We previously demonstrated magnetic nanoparticle (MNP) technology to remotely trigger adrenal hormone release by activating thermally sensitive ion channels. Here, we applied adrenal magnetothermal stimulation to probe stress-induced HPA axis changes. MNP and control nanoparticles were injected into the adrenal glands of outbred rats subjected to a tone-shock conditioning/extinction/recall paradigm. We measured MNP-triggered adrenal release before and after conditioning through physiologic (heart rate) and serum (epinephrine, corticosterone) markers. Aversive conditioning altered adrenal function, reducing corticosterone and blunting heart rate increases post-conditioning. MNP-based organ stimulation provides a novel approach to probing the function of HPA and other neuro-endocrine axes and could help elucidate changes across stress and disease models.

bioengineering↗

bioRxiv2020

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).

bioinformatics↗

bioRxiv2020

Herpes Simplex Virus Mistyping due to HSV-1 x HSV-2 Interspecies Recombination in Viral Gene Encoding Glycoprotein B

Human herpes simplex viruses (HSV) 1 and 2 are most often typed via molecular assays. Here we describe the first known case of HSV mistyping due to a previously undescribed HSV-1 x HSV-2 recombination event in UL27, the gene that encodes glycoprotein B. This is the first reported HSV interspecies recombination event impacting this gene, which is frequently used as a target for diagnostics and experimental therapeutics.

genomics↗

bioRxiv2019

Increased oral Epstein-Barr virus shedding with HIV-1 co-infection is due to a combination of B cell activation and impaired cellular immune control

Epstein-Barr virus (EBV) infection is transmitted by saliva and is a major cause of cancer in people living with HIV/AIDS as well as in the general population. To better understand the determinants of oral EBV shedding we evaluated the frequency and quantity of detectable EBV in the saliva in a prospective cohort study of 85 adults in Uganda, half of whom were co-infected with HIV-1. Participants were not receiving antiviral medications, and those with HIV-1 co-infection had a CD4+ T cell count >200 cells/mm3. Daily, self-collected oral swabs were collected over a 4-week period. Compared with HIV-1 uninfected participants, co-infected participants had an increased frequency of oral EBV shedding (IRR=1.27, 95% CI=1.10-1.47). To explain why EBV oral shedding is greater in HIV-1 co-infected participants, we developed a stochastic, mechanistic mathematical model that describes the dynamics of EBV, infected cells, and antiviral cellular immune responses within the tonsillar epithelium, and examined parameter-specific differences between individuals of different HIV-1 infection statuses. We fit the model to our observational data using Approximate Bayesian Computation. After fitting, model simulations showed high fidelity to daily oral shedding time-courses and matched key summary statistics. Examination of the model revealed that higher EBV loads in saliva are driven by B cell activation causing EBV lytic replication in the tonsils, in combination with a less effective EBV-specific cellular immune response. Thus, both these factors contribute to higher and more frequent EBV shedding in HIV-1 co-infected individuals compared to HIV-1 uninfected individuals. These conclusions were further validated by modelling daily oral EBV shedding in a 26-participant North American cohort. Our results provide insights into the determinants of EBV shedding and implicate B cell activation to be a potential therapeutic target to reduce EBV replication in HIV-1 co-infected individuals at high risk for EBV-related malignancies.\n\nAuthor summaryEpstein-Barr virus (EBV) is a ubiquitous infection worldwide. Infection with EBV is associated with the development of several kinds of cancer, including B cell lymphoma and nasopharyngeal carcinoma. Rates of EBV replication and disease are higher in individuals who are also infected with HIV-1. HIV-1 infection is associated with increased B cell activation, which is known to induce EBV reactivation, as well as immunodeficiency resulting from loss of T cells. However, whether these factors contribute to higher rates of EBV replication during co-infection, and by how much, was unknown. We analysed oral EBV shedding data in a cohort of adults from Uganda that were chronically infected with EBV. We found that participants that were HIV-1 infected were much more likely to have detectable quantities of EBV in their saliva. Also, when detected, the quantity of EBV present in the saliva was usually higher in HIV-1 infected participants. To better understand these findings, we developed a mathematical model to describe the dynamics of EBV, EBV-infected cells, and the cellular immune response within the tonsils. By rigorously matching our model to our participant data, we determined that high EBV loads in saliva are caused by high rates of infected B cell activation, as well as worse cellular immune control of EBV infection. These results provide an explanation of the impact of HIV-1 on EBV infection. Further, they suggest that strategies that suppress B cell activation may prevent EBV-related malignancy in people who are also infected with HIV-1.

pathology↗

bioRxiv2018

The impact of interspecies recombination on human herpes simplex virus evolution and host immune recognition

Among the most ubiquitous of human pathogens, HSV-1 and HSV-2 are distinct viral species that diverged about six million years ago. At least four ancient HSV-1 x HSV-2 interspecies recombination events have affected the HSV-2 genome, with recombinants and non-recombinants at each locus circulating today. Though interspecies recombination has occurred in the past, its importance in HSV evolution remains incompletely defined. Using 255 newly-sequenced and 219 existing HSV genome sequences, we comprehensively assessed interspecies recombination in HSV. The novel recombinants we identify demonstrate that the sizes and locations of interspecies recombination events in HSV-2 are more variable than previously appreciated. One novel recombinant arose in its current host, showing for the first time that interspecies recombination occurs in contemporary HSV populations. We also demonstrate that interspecies recombination affects T-cell recognition of HSV. Our findings indicate that interspecies recombination can significantly influence genetic variation in and host immunologic response to HSV-2.

genomics↗

bioRxiv2018

Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors

HIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect lower frequency alleles. However, the value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers Hydra and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low frequency mutations (1-20% frequency) associated with higher levels of drug resistance in 30/105 (29%) of protease-reverse transcriptase tests and 4/39 (10%) of integrase tests. Clinical follow-up of 69 individuals for a median of 674 days found no difference in rates of virological failure between individuals with and without low frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.\n\nImportanceGenotypic antiviral resistance testing for HIV is an essential component of the clinical microbiology and virology laboratory. Next-generation sequencing (NGS) has emerged as a powerful tool for the detection of low frequency sequence variants (allele frequencies <20%). Whether detecting these low frequency mutations in HIV contributes to improved patient health, however, remains unclear. We compared NGS to conventional Sanger sequencing for detecting resistance mutations for 144 HIV drug resistance tests submitted to our clinical virology laboratory and detected low frequency mutations in 24% of tests. Over approximately two years of follow-up for 69 patients for which we had access to electronic health records, no patients had virological failure due to antiviral resistance. Instead, virological failure was entirely explained by medication non-adherence. While larger studies are required, we suggest that detection of low frequency variants by NGS presents limited marginal clinical utility when compared to standard of care.

epidemiology↗

bioRxiv2018

Ultrasensitive capture of human herpes simplex virus genomes directly from clinical samples reveals extraordinarily limited evolution in cell culture

Herpes simplex viruses (HSV) are difficult to sequence due to their large DNA genome, high GC content, and the presence of repeats. To date, most HSV genomes have been recovered from culture isolates, raising concern that these genomes may not accurately represent circulating clinical strains. We report the development and validation of a DNA oligonucleotide hybridization panel to recover near complete HSV genomes at abundances up to 50,000-fold lower than previously reported. Using copy number information on herpesvirus and host DNA background via quantitative PCR, we developed a protocol for pooling for cost-effective recovery of more than 50 HSV-1 or HSV-2 genomes per MiSeq run. We demonstrate the ability to recover >99% of the HSV genome at >100X coverage in 72 hours at viral loads that allow whole genome recovery from latently-infected ganglia. We also report a new computational pipeline for rapid HSV genome assembly and annotation. Using the above tools and a series of 17 HSV-1-positive clinical swabs sent to our laboratory for viral isolation, we show limited evolution of HSV-1 during viral isolation in human fibroblast cells compared to the original clinical samples. Our data indicate that previous studies using low passage clinical isolates of herpes simplex viruses are reflective of the viral sequences present in the lesion and thus can be used in phylogenetic analyses. We also detect superinfection within a single sample with unrelated HSV-1 strains recovered from separate oral lesions in an immunosuppressed patient during a 2.5-week period, illustrating the power of direct-from-specimen sequencing of HSV.\n\nImportanceHerpes simplex viruses affect more than 4 billion people across the globe, constituting a large burden of disease. Understanding global diversity of herpes simplex viruses is important for diagnostics and therapeutics as well as cure research and tracking transmission among humans. To date, most HSV genomics has been performed on culture isolates and DNA swabs with high quantities of virus. We describe the development of wet-lab and computational tools that enable the accurate sequencing of near-complete genomes of HSV-1 and HSV-2 directly from clinical specimens at abundances >50,000-fold lower than previously sequenced and at significantly reduced cost. We use these tools to profile circulating HSV-1 strains in the community and illustrate limited changes to the viral genome during the viral isolation process. These techniques enable cost-effective, rapid sequencing of HSV-1 and HSV-2 genomes that will help enable improved detection, surveillance, and control of this human pathogen.

genomics↗

bioRxiv2018

Targeted mutation detection in breast cancer using MammaSeqTM

BackgroundBreast cancer is the most common invasive cancer among women worldwide. Next-generation sequencing (NGS) has revolutionized the study of cancer across research labs around the globe, however genomic testing in clinical settings remain limited. Advances in sequencing reliability, pipeline analysis, accumulation of relevant data, and the reduction of costs are rapidly increasing the feasibility of NGS-based clinical decision making.\n\nMethodsWe report the development of MammaSeq, a breast cancer specific NGS panel, targeting 79 genes and 1369 mutations, optimized for use in primary and metastatic breast cancer. To validate the panel, 46 solid tumor and 14 plasma circulating-free cfDNA samples were sequenced to a mean depth of 2311X and 1820 X respectively. Variants were called using Ion Torrent Suite 4.0 and annotated with cravat CHASM. CNVKit was used to call copy number variants in the solid tumor cohort. The oncoKB Precision Oncology Database was used to identify clinically actionable variants. ddPCR was used to validate select cfDNA mutations.\n\nResultsIn cohorts of 46 solid tumors and 14 cfDNA samples from patients with advanced breast cancer we identified 592 and 43 protein coding mutations. Mutations per sample in the solid tumor cohort ranged from 1 to 128 (median 3) and the cfDNA cohort ranged from 0 to 26 (median 2.5). Copy number analysis in the solid tumor cohort identified 46 amplifications and 35 deletions. We identified 26 clinically actionable variants (levels 1-3) annotated by OncoKB, distributed across 20 out of 46 cases (40%), in the solid tumor cohort. Allele frequencies of ESR1 and FOXA1 mutations correlated with CA.27.29 levels in patient matched blood draws.\n\nConclusionsIn solid tumors biopsies and cfDNA, MammaSeq detects clinicaly actionable mutations (oncoKB levels 1-3) in 22/46 (48%) solid tumors and in 4/14 (29%) of cfDNA samples. MammaSeq is a targeted panel suitable for clinically actionable mutation detection in breast cancer.

cancer biology↗