Transcriptional Analysis of the Guinea Pig Mucosal Immune Response to Intravaginal Infection with Herpes Simplex Virus Type 2.

Genital herpes infection in guinea pigs closely models human infection but tools for immune characterization are limited. Immunity to HSV infection at the vaginal epithelial surface was characterized in guinea pigs using PCR-based array analysis of vaginal swab samples. IFNγ was one of the most significantly upregulated genes throughout the infection and over 40% of genes with significantly altered expression were linked to IFNγ based on INTERFEROME analysis. IFNγ transcripts and biologically active IFNγ at the genital mucosa were confirmed by RTPCR and IFNγ reporter cells. Gene ontology analysis revealed activation of many biological processes related to genital immunity shared by humans and mice demonstrating the similarities of the local immune response to primary genital HSV-2 infection in guinea pigs and other established models. This transcription-based array will be useful for dissection of immunity during reactivation from latency, an infection outcome that is not well recapitulated by other animal models.

Development and utilization of a custom PCR array workflow: analysis of gene expression in mycoplasma genitalium and guinea pig (Cavia porcellus).

Transcriptome analysis is a powerful tool for evaluating molecular pathways central to maturation of specific biological processes and disease states. Recently, PCR-based arrays have supplemented microarray and RNA-seq methodologies for studying changes in gene expression levels. PCR arrays are a more cost efficient alternative, however commercially available assemblies are generally limited to only a few more widely researched species (e.g., rat, human, and mouse). Consequently, the investigation of emerging or under-studied species is hindered until such assays are created. To address this need, we present data documenting the success of a developed workflow with enhanced potential to create and validate novel RT-PCR arrays for underrepresented species with whole or partial genome annotation. Utilizing this enhanced workflow, we have achieved a success rate of 80 % for first-round designs for over 400 primer pairs. Of these, ~160 distinct targets were sequence confirmed. Proof of concept studies using two unique arrays, one targeting the pathogenic bacterium Mycoplasma genitalium and the other specific for the guinea pig (Cavia porcellus), allowed us to identify significant (P < 0.05) changes in mRNA expression validated by subsequent qPCR. This flexible and adaptable platform provides a valuable and cost-effective alternative for gene expression analysis.

Use of a Guinea pig-specific transcriptome array for evaluation of protective immunity against genital chlamydial infection following intranasal vaccination in Guinea pigs.

Guinea pigs have been used as a second animal model to validate putative anti-chlamydial vaccine candidates tested in mice. However, the lack of guinea pig-specific reagents has limited the utility of this animal model in Chlamydia sp. vaccine studies. Using a novel guinea pig-specific transcriptome array, we determined correlates of protection in guinea pigs vaccinated with Chlamydia caviae (C. caviae) via the intranasal route, previously reported by us and others to provide robust antigen specific immunity against subsequent intravaginal challenge. C. caviae vaccinated guinea pigs resolved genital infection by day 3 post challenge. In contrast, mock vaccinated animals continued to shed viable Chlamydia up to day 18 post challenge. Importantly, at day 80 post challenge, vaccinated guinea pigs experienced significantly reduced genital pathology - a sequelae of genital chlamydial infections, in comparison to mock vaccinated guinea pigs. Sera from vaccinated guinea pigs displayed antigen specific IgG responses and increased IgG1 and IgG2 titers capable of neutralizing GPIC in vitro. Th1-cellular/inflammatory immune genes and Th2-humoral associated genes were also found to be elevated in vaccinated guinea pigs at day 3 post-challenge and correlated with early clearance of the bacterium. Overall, this study provides the first evidence of guinea pig-specific genes involved in anti-chlamydial vaccination and illustrates the enhancement of the utility of this animal model in chlamydial pathogenesis.

Virus-specific immune memory at peripheral sites of herpes simplex virus type 2 (HSV-2) infection in guinea pigs.

Despite its importance in modulating HSV-2 pathogenesis, the nature of tissue-resident immune memory to HSV-2 is not completely understood. We used genital HSV-2 infection of guinea pigs to assess the type and location of HSV-specific memory cells at peripheral sites of HSV-2 infection. HSV-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. Memory B cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the HSV-specific antibody-secreting cells present at peripheral sites of HSV-2 infection represented persisting populations of plasma cells. The antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for HSV-2 glycoproteins and HSV-2 neutralizing antibodies. A vigorous IFN-γ-secreting T cell response developed in the spleen as well as the sites of HSV-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute HSV-2 infection. Additionally, populations of HSV-specific tissue-resident memory T cells were maintained at these sites and were readily detected up to 150 days post HSV-2 infection. Unlike the persisting plasma cells, HSV-specific memory T cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. Both HSV-specific CD4+ and CD8+ resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following HSV-2 infection. Together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of HSV-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of HSV-2 latency and spontaneous reactivation.

In vivo evaluation of antiviral efficacy against genital herpes using mouse and guinea pig models.

Both the guinea pig and mouse are important animal models for the study of genital herpes. The murine model has been used extensively to evaluate vaccines and antiviral agents by measuring the incidence of infection and the magnitude of viral replication; however, this model is limited with regard to distinguishing between candidate vaccines or treatments. In contrast, the guinea pig closely mimics human infection and provides an excellent model of both primary and recurrent genital herpes disease. This animal model is especially important in the study of viral transmission through the evaluation of latent viral reactivation and virus shedding into the genital tract. Here, we describe methodologies to determine viral infection, severity of primary disease, and quantification of primary viral replication in the genital tract for both the guinea pig and murine models of genital herpes. Additionally, we detail the evaluation of the onset of primary disease and progression to the day of death in the mouse model. Further, we summarize methods to assess the frequency of recurrences, frequency and magnitude of virus shedding, and latent viral load in the sensory nerve ganglia of the guinea pig.

The fibroblast growth factor 14·voltage-gated sodium channel complex is a new target of glycogen synthase kinase 3 (GSK3).

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na(+) (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na(+) currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.

A Vaxfectin(®)-adjuvanted HSV-2 plasmid DNA vaccine is effective for prophylactic and therapeutic use in the guinea pig model of genital herpes.

Here we describe studies in the guinea pig model of genital herpes to evaluate a novel plasmid DNA (pDNA) vaccine encoding the HSV-2 glycoprotein D and UL46 and UL47 genes encoding tegument proteins VP11/12 and VP 13/14 (gD2/UL46/UL47), formulated with a cationic lipid-based adjuvant Vaxfectin(®). Prophylactic immunization with Vaxfectin(®)-gD2/UL46/UL47 significantly reduced viral replication in the genital tract, provided complete protection against both primary and recurrent genital skin disease following intravaginal HSV-2 challenge, and significantly reduced latent HSV-2 DNA in the dorsal root ganglia compared to controls. We also examined the impact of therapeutic immunization of HSV-2 infected animals. Here, Vaxfectin(®)-gD2/UL46/UL47 immunization significantly reduced both the frequency of recurrent disease and viral shedding into the genital tract compared to controls. This novel adjuvanted pDNA vaccine has demonstrated both prophylactic and therapeutic efficacy in the guinea pig model of genital herpes and warrants further development.

Activity and the metabolic activation pathway of the potent and selective hepatitis C virus pronucleotide inhibitor PSI-353661.

PSI-353661, a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5'-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O(6)-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration.

Inhibition of hepatitis C virus replicon RNA synthesis by PSI-352938, a cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine.

PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine 5'-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 µM, respectively. The active 5'-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. In contrast, PSI-352938 did not inhibit the replication of hepatitis B virus or human immunodeficiency virus in vitro. PSI-352666 did not significantly affect the activity of human DNA and RNA polymerases. PSI-352938 and its cyclic phosphate metabolites did not affect the cyclic GMP-mediated activation of protein kinase G. Clearance studies using replicon cells demonstrated that PSI-352938 cleared cells of HCV replicon RNA and prevented replicon rebound. An additive to synergistic effect was observed when PSI-352938 was combined with other classes of HCV inhibitors, including alpha interferon, ribavirin, NS3/4A inhibitors, an NS5A inhibitor, and nucleoside/nucleotide and nonnucleoside inhibitors. Cross-resistance studies showed that PSI-352938 remained fully active against replicons containing the S282T or the S96T/N142T amino acid alteration. Replicons that contain mutations conferring resistance to various classes of nonnucleoside inhibitors also remained sensitive to inhibition by PSI-352938. PSI-352938 is currently being evaluated in a phase I clinical study in genotype 1-infected individuals.

Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor. Reduced RNA replication fitness and partial rescue by second-site mutations.

Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P'-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system.

We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.