Chk1 and the Host Cell DNA Damage Response as a Potential Antiviral Target in BK Polyomavirus Infection

The human BK polyomavirus (BKPyV) is latent in the kidneys of most adults, but can be reactivated in immunosuppressed states, such as following renal transplantation. If left unchecked, BK polyomavirus nephropathy (PyVAN) and possible graft loss may result from viral destruction of tubular epithelial cells and interstitial fibrosis. When coupled with regular post-transplant screening, immunosuppression reduction has been effective in limiting BKPyV viremia and the development of PyVAN. Antiviral drugs that are safe and effective in combating BKPyV have not been identified but would be a benefit in complementing or replacing immunosuppression reduction. The present study explores inhibition of the host DNA damage response (DDR) as an antiviral strategy. Immunohistochemical and immunofluorescent analyses of PyVAN biopsies provide evidence for stimulation of a DDR in vivo. DDR pathways were also stimulated in vitro following BKPyV infection of low-passage human renal proximal tubule epithelial cells. The role of Chk1, a protein kinase known to be involved in the replication stress-induced DDR, was examined by inhibition with the small molecule LY2603618 and by siRNA-mediated knockdown. Inhibition of Chk1 resulted in decreased replication of BKPyV DNA and viral spread. Activation of mitotic pathways was associated with the reduction in BKPyV replication. Chk1 inhibitors that are found to be safe and effective in clinical trials for cancer should also be evaluated for antiviral activity against BKPyV

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans

GM-CSF Increases Mucosal and Systemic Immunogenicity of an H1N1 Influenza DNA Vaccine Administered into the Epidermis of Non-Human Primates

Background: The recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a worldwide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques. Methodology/Principal Findings: Macaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particlemediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1-3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine. Conclusions/Significance: These results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skindelivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract. © 2010 Loudon et al

Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function

RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA’s cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs

The GM-CSF genetic adjuvant increases both the magnitude and breadth of mucosal T cell responses elicited in the lungs and guts following PMED HA DNA vaccination.

<p>HA-specific T cell responses in the (<b>A</b>) lung and (<b>B</b>) gut mucosa of macaques were determined by IFN-γ ELISpot assay 4–11 weeks after the final vaccination with HA DNA (solid bars) or HA DNA+GM-CSF (hatched bars). Individual bars represent the peak number of HA-specific IFN-γ T cells detected in the jejunum and lung tissue of individual animals 4–11 weeks after the final vaccination. (<b>C</b>) Breadth of the IFN-γ T cell response in PBMC, lung, and gut. HA-specific T cell responses in the indicated tissues were measured using a standard IFN-γ ELISpot assay with 6 individual pools of overlapping peptides (11 amino acid overlaps, 103 15-mers per pool) comprising the entire amino acid sequence of the influenza A/New Caledonia/20/99 HA protein. The percent contribution of each peptide-pool specific response to the total response was determined by dividing the mean number of IFN-γ spot forming cells (SFC) measured against each individual peptide pool by the sum of the response against all peptide pools. Results represent the average of 2 time-points tested after the 3^rd DNA dose (weeks 19 and 23). *Measurement below positive threshold level for the assay.</p

Induction of mucosal antibody responses in the respiratory tract of PMED HA DNA vaccinated macaques.

<p>Bronchoalveolar lavage fluid (BALF) and tracheal swab samples were collected from macaques prior to immunization and again at 4 weeks following each boosting dose of the HA DNA vaccine, ± rhGM-CSF. IgG antibody responses in BALF (<b>A</b>) and IgA antibody responses in tracheal swabs (<b>B</b>) against the A/New Caledonia/20/99 hemagglutinin protein were detected by ELISA. Data are reported as the O.D. measured at 450 nm for each sample, diluted 1∶20 in PBS, from individual immunized animals.</p