Inference for Nonlinear Epidemiological Models Using Genealogies and Time Series

Phylodynamics - the field aiming to quantitatively integrate the ecological and evolutionary dynamics of rapidly evolving populations like those of RNA viruses – increasingly relies upon coalescent approaches to infer past population dynamics from reconstructed genealogies. As sequence data have become more abundant, these approaches are beginning to be used on populations undergoing rapid and rather complex dynamics. In such cases, the simple demographic models that current phylodynamic methods employ can be limiting. First, these models are not ideal for yielding biological insight into the processes that drive the dynamics of the populations of interest. Second, these models differ in form from mechanistic and often stochastic population dynamic models that are currently widely used when fitting models to time series data. As such, their use does not allow for both genealogical data and time series data to be considered in tandem when conducting inference. Here, we present a flexible statistical framework for phylodynamic inference that goes beyond these current limitations. The framework we present employs a recently developed method known as particle MCMC to fit stochastic, nonlinear mechanistic models for complex population dynamics to gene genealogies and time series data in a Bayesian framework. We demonstrate our approach using a nonlinear Susceptible-Infected-Recovered (SIR) model for the transmission dynamics of an infectious disease and show through simulations that it provides accurate estimates of past disease dynamics and key epidemiological parameters from genealogies with or without accompanying time series data

A Novel Approach of Identifying Immunodominant Self and Viral Antigen Cross-Reactive T Cells and Defining the Epitopes They Recognize

Infection and vaccination can lead to activation of autoreactive T cells, including the activation of cross-reactive T cells. However, detecting these cross-reactive T cells and identifying the non-self and self-antigen epitopes is difficult. The current study demonstrates the utility of a novel approach that effectively accomplishes both. We utilized surface expression of CD38 on newly activated CD4 memory T cells as a strategy to identify type 1 diabetes associated autoreactive T cells activated by influenza vaccination in healthy subjects. We identified an influenza A matrix protein (MP) specific CD4+ T cell clone that cross-recognizes an immunodominant epitope from Glutamic Acid Decarboxylase 65 (GAD65) protein. The sequences of the MP and GAD65 peptides are rather distinct, with only 2 identical amino acids within the HLA-DR binding region. This result suggests that activation of autoreactive T cells by microbial infection under certain physiological conditions can occur amongst peptides with minimum amino acid sequence homology. This novel strategy also provides a new research pathway in which to examine activation of autoreactive CD4+ T cells after vaccination or natural infection

A Flexible Framework for the Creation of Narrative-Centered Tools

To better support the creation of narrative-centered tools, developers need a flexible framework to integrate, catalog, select, and reuse narrative models. Computational models of narrative enable the creation of software tools to aid narrative processing, analysis, and generation. Narrative-centered tools explicitly or implicitly embody one or more models of narrative by their definition. However, narrative model creation is often expensive and difficult with no guaranteed benefit to the end system. This paper describes our preliminary approach towards creating the SONNET narrative framework, a flexible framework to integrate, catalog, select, and reuse narrative models, thereby lowering development costs and improving benefits from each model. The framework includes a lightweight ontology language for the definition of key terms and interrelationships among them. The framework specifies model metadata to allow developers to discover and understand models more readily. We discuss the structure of this framework and ongoing development incorporating narrative models

Vascular E-selectin expression correlates with CD8 lymphocyte infiltration and improved outcome in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-linked skin cancer. While CD8 lymphocyte infiltration into the tumor is strongly correlated with improved survival, these cells are absent or sparse in most MCCs. We investigated whether specific mechanisms of T-cell migration may be commonly disrupted in MCC tumors with poor CD8 lymphocyte infiltration. Intratumoral vascular E-selectin, critical for T-cell entry into skin, was downregulated in the majority (52%) of MCCs (n=56), and its loss was associated with poor intratumoral CD8 lymphocyte infiltration (p<0.05; n=45). Importantly, survival was improved in MCC patients whose tumors had higher vascular E-selectin expression (p<0.05). Local nitric oxide (NO) production is one mechanism of E-selectin downregulation and it can be tracked by quantifying nitrotyrosine, a stable biomarker of NO-induced reactive nitrogen species (RNS). Indeed, increasing levels of nitrotyrosine within MCC tumors were associated with low E-selectin expression (p<0.05; n=45) and decreased CD8 lymphocyte infiltration (p<0.05, n=45). These data suggest that one mechanism of immune evasion in MCC may be restriction of T cell entry into the tumor. Existing therapeutic agents that modulate E-selectin expression and/or RNS generation may restore T cell entry and could potentially synergize with other immune-stimulating therapies

Acute Infection and Subsequent Subclinical Reactivation of Herpes Simplex Virus 2 after Vaginal Inoculation of Rhesus Macaques.

Herpes simplex virus 2 (HSV-2) is a common sexually transmitted infection with a highly variable clinical course. Many infections quickly become subclinical, with episodes of spontaneous virus reactivation. To study host-HSV-2 interactions, an animal model of subclinical HSV-2 infection is needed. In an effort to develop a relevant model, rhesus macaques (RM) were inoculated intravaginally with two or three HSV-2 strains (186, 333, and/or G) at a total dose of 1 × 107 PFU of HSV-2 per animal. Infectious HSV-2 and HSV-2 DNA were consistently shed in vaginal swabs for the first 7 to 14 days after each inoculation. Proteins associated with wound healing, innate immunity, and inflammation were significantly increased in cervical secretions immediately after HSV-2 inoculation. There was histologic evidence of acute herpesvirus pathology, including acantholysis in the squamous epithelium and ballooning degeneration of and intranuclear inclusion bodies in epithelial cells, with HSV antigen in mucosal epithelial cells and keratinocytes. Further, an intense inflammatory infiltrate was found in the cervix and vulva. Evidence of latent infection and reactivation was demonstrated by the detection of spontaneous HSV-2 shedding post-acute inoculation (102 to 103 DNA copies/swab) in 80% of RM. Further, HSV-2 DNA was detected in ganglia in most necropsied animals. HSV-2-specifc T-cell responses were detected in all animals, although antibodies to HSV-2 were detected in only 30% of the animals. Thus, HSV-2 infection of RM recapitulates many of the key features of subclinical HSV-2 infection in women but seems to be more limited, as virus shedding was undetectable more than 40 days after the last virus inoculation.IMPORTANCE Herpes simplex virus 2 (HSV-2) infects nearly 500 million persons globally, with an estimated 21 million incident cases each year, making it one of the most common sexually transmitted infections (STIs). HSV-2 is associated with increased human immunodeficiency virus type 1 (HIV-1) acquisition, and this risk does not decline with the use of antiherpes drugs. As initial acquisition of both HIV and HSV-2 infections is subclinical, study of the initial molecular interactions of the two agents requires an animal model. We found that HSV-2 can infect RM after vaginal inoculation, establish latency in the nervous system, and spontaneously reactivate; these features mimic some of the key features of HSV-2 infection in women. RM may provide an animal model to develop strategies to prevent HSV-2 acquisition and reactivation

Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation

Cytotoxic CD8+ T cells play a critical role in controlling herpes simplex virus (HSV) infection and reactivation. However, little is known about the spatiotemporal dynamics of CD8+ T cells during HSV lesion evolution or about their involvement in immune surveillance after lesion resolution. Using quantum dot–conjugated peptide–major histocompatibility complex multimers, we investigated the in vivo localization of HSV-2–specific CD8+ T cells in sequential biopsies of human genital skin during acute, resolving, and healed stages of HSV-2 reactivation. Our studies revealed that functionally active CD8+ T cells selectively infiltrated to the site of viral reactivation. After lesion healing in concert with complete reepithelialization and loss of HSV DNA from skin biopsies, HSV-2–specific CD8+ T cells persisted for more than two months at the dermal–epidermal junction, adjacent to peripheral nerve endings. In two out of the six sequentially studied individuals, HSV-2 DNA reappeared in clinically and histologically normal–appearing skin. Detection of viral DNA was accompanied by increased numbers of both HSV-specific and total CD8+ T cells in the dermis. These findings indicate that the frequency and clinical course of HSV-2 reactivation in humans is influenced by virus-specific CD8+ T cells that persist in peripheral mucosa and genital skin after resolution of herpes lesions

CD4 T-cell memory responses to viral infections of humans show pronounced immunodominance independent of duration or viral persistence

Little is known concerning immunodominance within the CD4 T-cell response to viral infections and its persistence into longterm memory. We tested CD4 T-cell reactivity against each viral protein in persons immunized with vaccinia virus (VV), either recently or more than 40 years ago, as a model self-limited v

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3’ scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets. IMPORTANCE Most people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.</p

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3’ scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets. IMPORTANCE Most people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.</p

A Naturally Selected Dimorphism within the HLA-B44 Supertype Alters Class I Structure, Peptide Repertoire, and T Cell Recognition

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the α2 helix (B*4402 Asp156→B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this “minimal” mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire