Joshua T. Schiffer

Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections

ABSTRACT Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches.

Standard-dose and high-dose daily antiviral therapy for short episodes of genital HSV-2 reactivation: three randomised, open-label, cross-over trials

BACKGROUND Skin and mucosal herpes simplex virus type 2 (HSV-2) shedding predominantly occurs in short subclinical episodes. We assessed whether standard-dose or high-dose antiviral therapy reduces the frequency of such shedding. METHODS HSV-2-seropositive, HIV-seronegative people were enrolled at the University of Washington Virology Research Clinic (WA, USA). We did three separate but complementary open-label cross-over studies comparing no medication with aciclovir 400 mg twice daily (standard-dose aciclovir), valaciclovir 500 mg daily (standard-dose valaciclovir) with aciclovir 800 mg three times daily (high-dose aciclovir), and standard-dose valaciclovir with valaciclovir 1 g three times daily (high-dose valaciclovir). The allocation sequence was generated by a random number generator. Study drugs were supplied in identical, numbered, sealed boxes. Study periods lasted 4-7 weeks, separated by 1 week wash-out. Participants collected genital swabs four times daily for quantitative HSV DNA PCR. Clinical data were masked from laboratory personnel. The primary endpoint was within-person comparison of shedding rate in each study group. Analysis was per protocol. The trials are registered at ClinicalTrials.gov (NCT00362297, NCT00723229, NCT01346475). RESULTS Of 113 participants randomised, 90 were eligible for analysis of the primary endpoint. Participants collected 23 605 swabs; 1272 (5·4%) were HSV-positive. The frequency of HSV shedding was significantly higher in the no medication group (n=384, 18·1% of swabs) than in the standard-dose aciclovir group (25, 1·2%; incidence rate ratio [IRR] 0·05, 95% CI 0·03-0·08). High-dose aciclovir was associated with less shedding than standard-dose valaciclovir (198 [4·2%] vs 209 [4·5%]; IRR 0·79, 95% CI 0·63-1·00). Shedding was less frequent in the high-dose valaciclovir group than in the standard-dose valaciclovir group (164 [3·3%] vs 292 [5·8%]; 0·54, 0·44-0·66). The number of episodes per person-year did not differ significantly for standard-dose valaciclovir (22·6) versus high-dose aciclovir (20·2; p=0·54), and standard-dose valaciclovir (14·9) versus high-dose valaciclovir (16·5; p=0·34), but did for no medication (28·7) and standard-dose aciclovir (10·0; p=0·001). Median episode duration was longer for no medication than for standard-dose aciclovir (13 h vs 7 h; p=0·01) and for standard-dose valaciclovir than for high-dose valaciclovir (10 h vs 7 h; p=0·03), but did not differ significantly between standard-dose valaciclovir and high-dose aciclovir (8 h vs 8 h; p=0·23). Likewise, maximum log(10) copies of HSV detected per mL was higher for no medication than for standard dose aciclovir (3·3 vs 2·9; p=0·02), and for standard-dose valaciclovir than for high-dose valaciclovir (2·5 vs 3·0; p=0·001), but no significant difference was recorded for standard-dose valaciclovir versus high-dose aciclovir (2·7 vs 2·8; p=0·66). 80% of episodes were subclinical in all study groups. Except for a higher frequency of headaches with high-dose valaciclovir (n=13, 30%) than with other regimens, all regimens were well tolerated. INTERPRETATION Short bursts of subclinical genital HSV reactivation are frequent, even during high-dose antiherpes therapy, and probably account for continued transmission of HSV during suppressive antiviral therapy. More potent antiviral therapy is needed to eliminate HSV transmission. FUNDING NIH. Valaciclovir was provided for trial 3 for free by GlaxoSmithKline.

Mucosal host immune response predicts the severity and duration of herpes simplex virus-2 genital tract shedding episodes

Herpes simplex virus-2 (HSV-2) shedding episodes in humans vary markedly in duration and virologic titer within an infected person over time, an observation that is unexplained. To evaluate whether host or virological factors more closely accounted for this variability, we combined measures of viral replication and CD8+ lymphocyte density in genital biopsies, with a stochastic mathematical model of HSV-2 infection. Model simulations reproduced quantities of virus and duration of shedding detected in 1,003 episodes among 386 persons. In the simulations, local CD8+ lymphocyte density in the mucosa at episode onset predicted peak HSV DNA copy number and whether genital lesions or subclinical shedding occurred. High density of CD8+ T cells in the mucosa correlated with decreased infected cell lifespan and fewer infected epithelial cells before episode clearance. If infected cell lifespan increased by 15 min because of CD8+ lymphocyte decay, then there was potential for a thousandfold increase in the number of infected cells. The model suggests that the rate of containment of infected cells by the peripheral mucosal immune system is the major driver of duration and severity of HSV-2 reactivation in the immunocompetent host.

Frequent Release of Low Amounts of Herpes Simplex Virus from Neurons: Results of a Mathematical Model

A stochastic mathematical model suggests that very low numbers of herpes simplex virus 2 particles are released frequently into the genital tract in infected individuals. Insights from Modeling Herpes Virus Behavior One variety of the herpes simplex virus (HSV-2) infects 45 million people worldwide, often causing ulcers in the genital region, and increases the acquisition and transmission of HIV. Herpes is passed from person to person through sexual contact. After the initial infection, the herpes virus resides in the cell bodies of neurons that innervate the genital tract, sometimes causing recurrences of the painful genital blisters. With newer, highly sensitive methods of detecting the virus by the polymerase chain reaction, the pattern of HSV-2 shedding in the genital tract was unexpectedly revealed to include frequent, intermittent episodes of variable amounts of virus, not just the periodic larger episodes that had been assumed to cause the sporadic recurrence of genital ulcers. This finding has been of concern for several reasons: The likelihood of transmitting the virus to a sexual partner is higher if herpes virus is often present in the genital tract of infected people. And worse, most episodes of release of the small amounts of virus are not accompanied by any symptoms, so infected individuals are often unaware of their own infectious state. A clearer understanding of how virus release into the genital tract is controlled and when it causes an active ulcer is needed to develop better ways to combat this common infection. Available treatments, mainly antiviral agents, can only limit spread and accelerate healing but do not completely eliminate asymptomatic shedding or transmission. Because informative experiments are difficult to do in humans, Schiffer et al. developed a mathematical model of HSV-2 infection of mucosal tissue. Their model—which incorporated virus release to the genital mucosal tissue, infection of epithelial cells, replication of virus within epithelial cells, and an immune response—faithfully replicated shedding episode data from anogenital swabs from 89 patients and from lesions of 15 more patients. It predicted that infected epithelial cells can generate hundreds of times more virus than the infected neurons, accounting for detectable shedding episodes. A stochastic version of the model predicted variability in the patterns of herpes infection seen in patients with respect to the percentage of time spent shedding and the lesion diameter. The model also suggested that there are many clinical episodes of viral shedding that cannot be detected by current methods. Most important, the analysis led the authors to conclude that neurons releasing only a few copies of herpes virus each day can cause the characteristic periodic outbreaks of painful ulcers and that it is the amount of virus released per unit time, not the frequency of release, that controls how often detectable virus appears in the genital tract. The authors used both patient data and the cellular biology of herpes infection to build an informative model that has revealed a control point for a key determinant of viral infectivity, suggesting a vulnerable target for therapeutic intervention. Herpes simplex virus 2 (HSV-2), a sexually transmitted infection, is the leading cause of genital ulcers worldwide. Infection is lifelong and is characterized by repeated asymptomatic and symptomatic episodes of virus shedding that are initiated when virus is released from neurons into the genital tract. The pattern of HSV-2 release from neurons, which harbor the virus, into the genital tract is poorly understood. We fit a mathematical model of HSV-2 pathogenesis to curves generated from daily quantification of HSV in mucosal swabs from patients with herpetic genital ulcers. We used virologic parameters derived from model fitting for stochastic model simulations. These simulations reproduced previously documented estimates for shedding frequency and herpetic lesion diameter and frequency. The most realistic model output occurred when we assumed that the amount of virus shed from neurons daily was minimal. In our simulations, small changes in the average total quantity of HSV-2 released from neurons influenced the frequency of detectable shedding, whereas changes in the frequency of HSV-2 neuronal release had little effect. Frequent HSV-2 shedding episodes in humans are explained by nearly constant release of small numbers of viruses from neurons that terminate in the genital tract.

Timing and severity of community acquired respiratory virus infections after myeloablative versus non-myeloablative hematopoietic stem cell transplantation

Respiratory virus infections are important causes of morbidity and mortality after hematopoietic cell transplantation. Their clinical course can be severe with progression to lower respiratory tract infection, co-infection with serious pulmonary co-pathogens, and high mortality. The findings of this retrospective cohort study indicate that viral lower respiratory tract infection during the first 100 days after hematopoietic cell transplantation was less common among patients undergoing non-myeloablative conditioning regimens than in those receiving myeloablative conditioning, despite a similar overall rate of acquisition. Background Respiratory virus infections are important causes of morbidity and mortality after hematopoietic cell transplantation. Their clinical course can be severe with progression to lower respiratory tract infection, co-infection with serious pulmonary co-pathogens, and high mortality. Non-myeloablative conditioning regimens achieve engraftment without eradication of host hematopoietic cells, which potentially allows for protection against infections commonly seen in hematopoietic cell transplantation patients treated with standard intensity conditioning regimens. Design and Methods We performed a retrospective cohort study to measure the incidence and severity of parainfluenza types 1–4, influenza (A and B), respiratory syncitial virus and human rhinovirus disease in myeloablative versus non-myeloablative versus autologous hematopoietic cell transplantation patients. Results The incidences of all respiratory virus infections were similar in the non-myeloablative and myeloablative cohorts but less in the autologous cohort (33/420 [7.9%], 150/1593 [9.4%], and 37/751 [4.9%], respectively, p<0.0001). However, respiratory virus lower tract infections were significantly less common during the first 100 days after transplantation in non-myeloablative patients compared to myeloablative and autologous patients (1/420 [0.2%], 34/1593 [2.1%] and 16/751 [2.1%], respectively, p=0.005. Respiratory virus lower tract infection had high co-infection and attributable mortality rates. Conclusions Respiratory virus lower tract infection during the first 100 days after hematopoietic cell transplantation was less common in persons receiving non-myeloablative conditioning regimens compared to myeloablative conditioning, despite a similar overall rate of acquisition.

AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

Rapid host immune response and viral dynamics in herpes simplex virus-2 infection.

Herpes simplex virus-2 (HSV-2) is periodically shed throughout the human genital tract. Although a high viral load correlates with the development of genital ulcers, shedding also commonly occurs even when ulcers are absent, allowing for silent transmission during coitus and contributing to high seroprevalence of HSV-2 worldwide. Frequent viral reactivation occurs within ganglia despite diverse and complementary host and viral mechanisms that predispose toward latency, suggesting that viral replication may be constantly occurring in a small minority of neurons at these sites. Within genital mucosa, the in vivo expansion and clearance rates of HSV-2 are extremely rapid. Resident dendritic cells and memory HSV-2 specific T cells persist at prior sites of genital tract reactivation and, in conjunction with prompt innate recognition of infected cells, lead to rapid containment of infected cells. The fact that immune responses usually control viral replication in genital skin before lesions develop provides hope that enhancing such responses could lead to effective vaccines and immunotherapies.

Rapid localized spread and immunologic containment define Herpes simplex virus-2 reactivation in the human genital tract

Herpes simplex virus-2 (HSV-2) is shed episodically, leading to occasional genital ulcers and efficient transmission. The biology explaining highly variable shedding patterns, in an infected person over time, is poorly understood. We sampled the genital tract for HSV DNA at several time intervals and concurrently at multiple sites, and derived a spatial mathematical model to characterize dynamics of HSV-2 reactivation. The model reproduced heterogeneity in shedding episode duration and viral production, and predicted rapid early viral expansion, rapid late decay, and wide spatial dispersion of HSV replication during episodes. In simulations, HSV-2 spread locally within single ulcers to thousands of epithelial cells in <12 hr, but host immune responses eliminated infected cells in <24 hr; secondary ulcers formed following spatial propagation of cell-free HSV-2, allowing for episode prolongation. We conclude that HSV-2 infection is characterized by extremely rapid virological growth and containment at multiple contemporaneous sites within genital epithelium. DOI: http://dx.doi.org/10.7554/eLife.00288.001

The Kinetics of Mucosal Herpes Simplex Virus-2 Infection in Humans: Evidence for Rapid Viral-Host Interactions

BACKGROUND Herpes simplex virus type 2 (HSV-2) reactivations in the genital tract are responsible for mucocutaneous lesions and transmission and manifest as discrete shedding episodes. METHODS We analyzed duration, peak copy number, and expansion and decay rates of 1020 shedding episodes in 531 immunocompetent HSV-2-seropositive persons from whom daily swabs of genital secretions were collected. RESULTS Viral quantity varied by as much as a multiple of 10 million in a single person over time. Peak episode copy number was distributed approximately evenly from 10(3) through 10(8) HSV DNA copies/mL. Median rate of increase was 10(7.6) HSV DNA copies/day during the first 12 hours of an episode and 10(5) copies/d from episode initiation to peak. These values depended only moderately on episode duration. Median decay rate was -10(6.2) HSV DNA copies/d during the final 12 hours of an episode and -10(3.6) copies/d from peak to termination. Episodes lasted a median of 3 days (interquartile range, 1-8 days). Prolonged (>5 days) episodes were associated with nonmonotonic decay. CONCLUSIONS HSV-2 shedding episodes are notable for rapid expansion and decay and extreme heterogeneity of duration and viral production. The net effect of these dynamic episodes is frequent shedding at high copy numbers.

Predictors of Hepatitis B Cure Using Gene Therapy to Deliver DNA Cleavage Enzymes: A Mathematical Modeling Approach

Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA), the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which will in turn provide vital information for dose selection for potential curative trials in animals and ultimately humans.