Kening Wang

An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.

Laser-Capture Microdissection: Refining Estimates of the Quantity and Distribution of Latent Herpes Simplex Virus 1 and Varicella-Zoster Virus DNA in Human Trigeminal Ganglia at the Single-Cell Level

ABSTRACT There remains uncertainty and some controversy about the percentages and types of cells in human sensory nerve ganglia that harbor latent herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) DNA. We developed and validated laser-capture microdissection and real-time PCR (LCM/PCR) assays for the presence and copy numbers of HSV-1 gG and VZV gene 62 sequences in single cells recovered from sections of human trigeminal ganglia (TG) obtained at autopsy. Among 970 individual sensory neurons from five subjects, 2.0 to 10.5% were positive for HSV-1 DNA, with a median of 11.3 copies/positive cell, compared with 0.2 to 1.5% of neurons found to be positive by in situ hybridization (ISH) for HSV-1 latency-associated transcripts (LAT), the classical surrogate marker for HSV latency. This indicates a more pervasive latent HSV-1 infection of human TG neurons than originally thought. Combined ISH/LCM/PCR assays revealed that the majority of the latently infected neurons do not accumulate LAT to detectable levels. We detected VZV DNA in 1.0 to 6.9% of individual neurons from 10 subjects. Of the total 1,722 neurons tested, 4.1% were VZV DNA positive, with a median of 6.9 viral genomes/positive cell. After removal by LCM of all visible neurons on a slide, all surrounding nonneuronal cells were harvested and assayed: 21 copies of HSV-1 DNA were detected in ∼5,200 nonneuronal cells, while nine VZV genomes were detected in ∼14,200 nonneuronal cells. These data indicate that both HSV-1 and VZV DNAs persist in human TG primarily, if not exclusively, in a moderate percentage of neuronal cells.

Comparative Efficacy and Immunogenicity of Replication-Defective, Recombinant Glycoprotein, and DNA Vaccines for Herpes Simplex Virus 2 Infections in Mice and Guinea Pigs

ABSTRACT Many candidate vaccines are effective in animal models of genital herpes simplex virus type 2 (HSV-2) infection. Among them, clinical trials showed moderate protection from genital disease with recombinant HSV-2 glycoprotein D (gD2) in alum-monophosphoryl lipid A adjuvant only in HSV women seronegative for both HSV-1 and HSV-2, encouraging development of additional vaccine options. Therefore, we undertook direct comparative studies of the prophylactic and therapeutic efficacies and immunogenicities of three different classes of candidate vaccines given in four regimens to two species of animals: recombinant gD2, a plasmid expressing gD2, and dl5-29, a replication-defective strain of HSV-2 with the essential genes UL5 and UL29 deleted. Both dl5-29 and gD2 were highly effective in attenuating acute and recurrent disease and reducing latent viral load, and both were superior to the plasmid vaccine alone or the plasmid vaccine followed by one dose of dl5-29. dl5-29 was also effective in treating established infections. Moreover, latent dl5-29 virus could not be detected by PCR in sacral ganglia from guinea pigs vaccinated intravaginally. Finally, dl5-29 was superior to gD2 in inducing higher neutralizing antibody titers and the more rapid accumulation of HSV-2-specific CD8+ T cells in trigeminal ganglia after challenge with wild-type virus. Given its efficacy, its defectiveness for latency, and its ability to induce rapid, virus-specific CD8+-T-cell responses, the dl5-29 vaccine may be a good candidate for early-phase human trials.

Painful Pathways Induced by TLR Stimulation of Dorsal Root Ganglion Neurons

We hypothesize that innate immune signals from infectious organisms and/or injured tissues may activate peripheral neuronal pain signals. In this study, we demonstrated that TLRs 3, 7, and 9 are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proinflammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP-10), IL-1α, IL-1β, and PGE2, which have previously been shown to augment pain. Further, TLR ligands upregulated the expression of a nociceptive receptor, transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1-expressing DRGNs. Using a tumor-induced temperature sensitivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive oligodeoxynucleotide, blocked tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce nerve pain.

Identification of Viral MicroRNAs Expressed in Human Sacral Ganglia Latently Infected with Herpes Simplex Virus 2

ABSTRACT Deep sequencing of small RNAs isolated from human sacral ganglia latently infected with herpes simplex virus 2 (HSV-2) was used to identify HSV-2 microRNAs (miRNAs) expressed during latent infection. This effort resulted in the identification of five distinct HSV-2 miRNA species, two of which, miR-H3/miR-I and miR-H4/miR-II, have been previously reported. Three novel HSV-2 miRNAs were also identified, and two of these, miR-H7 and miR-H9, are derived from the latency-associated transcript (LAT) and are located antisense to the viral transcript encoding transactivator ICP0. A third novel HSV-2 miRNA, miR-H10, is encoded within the unique long (UL) region of the genome, 3′ to the UL15 open reading frame, and is presumably excised from a novel, latent HSV-2 transcript distinct from LAT.

Genome Sequencing and Analysis of Geographically Diverse Clinical Isolates of Herpes Simplex Virus 2

ABSTRACT Herpes simplex virus 2 (HSV-2), the principal causative agent of recurrent genital herpes, is a highly prevalent viral infection worldwide. Limited information is available on the amount of genomic DNA variation between HSV-2 strains because only two genomes have been determined, the HG52 laboratory strain and the newly sequenced SD90e low-passage-number clinical isolate strain, each from a different geographical area. In this study, we report the nearly complete genome sequences of 34 HSV-2 low-passage-number and laboratory strains, 14 of which were collected in Uganda, 1 in South Africa, 11 in the United States, and 8 in Japan. Our analyses of these genomes demonstrated remarkable sequence conservation, regardless of geographic origin, with the maximum nucleotide divergence between strains being 0.4% across the genome. In contrast, prior studies indicated that HSV-1 genomes exhibit more sequence diversity, as well as geographical clustering. Additionally, unlike HSV-1, little viral recombination between HSV-2 strains could be substantiated. These results are interpreted in light of HSV-2 evolution, epidemiology, and pathogenesis. Finally, the newly generated sequences more closely resemble the low-passage-number SD90e than HG52, supporting the use of the former as the new reference genome of HSV-2. IMPORTANCE Herpes simplex virus 2 (HSV-2) is a causative agent of genital and neonatal herpes. Therefore, knowledge of its DNA genome and genetic variability is central to preventing and treating genital herpes. However, only two full-length HSV-2 genomes have been reported. In this study, we sequenced 34 additional HSV-2 low-passage-number and laboratory viral genomes and initiated analysis of the genetic diversity of HSV-2 strains from around the world. The analysis of these genomes will facilitate research aimed at vaccine development, diagnosis, and the evaluation of clinical manifestations and transmission of HSV-2. This information will also contribute to our understanding of HSV evolution.

Diverse Herpes Simplex Virus Type 1 Thymidine Kinase Mutants in Individual Human Neurons and Ganglia

ABSTRACT Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations of tk mutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSV tk mutants in animal neurons is impaired. Here we report the detection of mutated HSV tk sequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1 tk open reading frames from ganglia were amplified by PCR, cloned, and sequenced. tk mutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1 tk mutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSV tk mutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected with tk mutant virus alone. These data suggest that diverse tk mutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.

The 2.2-Kilobase Latency-Associated Transcript of Herpes Simplex Virus Type 2 Does Not Modulate Viral Replication, Reactivation, or Establishment of Latency in Transgenic Mice

ABSTRACT To better understand the mechanisms responsible for the observed effects of deletions in the promoter region of the latency-associated transcript (LAT) gene in impairing herpes simplex virus (HSV) reactivation, we generated mice transgenic for a 5.5-kb HSV type 2 (HSV-2) genomic fragment spanning the major LAT, along with the LAT promoter and flanking regions, in the C57BL/6 background. The mice expressed abundant 2.2-kb major LATs in trigeminal ganglia (TG) and other tissues. The effects of the transgene on HSV-2 infection, latency, and reactivation were assessed. When infected with wild-type (WT) HSV-2 or its LAT promoter deletion (LAT−) mutant, primary lung fibroblast lines established from normal C57BL/6 and transgenic mice supported virus growth equally well. The replication of these viruses in the mouse eye and their spread to TG and brains were similar. The quantities of latent viral DNA in TG of transgenic and normal mice, as determined by real-time PCR, were comparable. UV light-induced reactivation of the LAT− mutant from transgenic mice (0 to 7%) was no more frequent than that from normal mice (0 to 14%), while WT virus was reactivated from 13 to 54% of normal mice and 22 to 54% of transgenic mice. The cumulative data indicate that, when expressed transgenically, the HSV-2 major LAT cannot influence HSV-2 infection or latency and cannot complement the defect in reactivation of the LAT− mutant. These results imply that the phenotype of reduced reactivation associated with the LAT− mutant is related to a function encoded in the LAT promoter but not to the major LAT itself.

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

ABSTRACT We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

Topical Herpes Simplex Virus 2 (HSV-2) Vaccination with Human Papillomavirus Vectors Expressing gB/gD Ectodomains Induces Genital-Tissue-Resident Memory CD8+ T Cells and Reduces Genital Disease and Viral Shedding after HSV-2 Challenge

ABSTRACT No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8+ T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8+ T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. IMPORTANCE Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.