Most pathogens initiate infection via the mucosa, therefore delivery of vaccines directly to the mucosa is likely to be advantageous for stimulating protective immunity at the site of entry. PilVax is a novel mucosal vaccine platform that harnesses Lactococcus lactis bacteria engineered to stably express multiple copies of vaccine peptide antigens within pili, hair-like structures which extend from the cell wall. This strategy elicited systemic and mucosal antibody responses to a model antigen after intranasal immunization, but has not been tested for its capacity to stimulate protective mucosal immunity. A well-characterized linear B-cell epitope, D3 (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) , from the fibronectin-binding protein A of Staphylococcus aureus was successfully introduced into PilVax and delivered intranasally to mice. Specific antipeptide immunoglobulin (Ig) G and IgA antibodies were detected in the serum and respiratory mucosa of vaccinated mice. Responses to the major pilus backbone protein Spy0128 were also assessed; robust antibody responses to this antigen were generated both systemically and in the respiratory and intestinal mucosa. Mice were challenged intranasally with the mouse-adapted S. aureus JSNZ strain and the S. aureus load quantified 7 days after challenge. Unexpectedly, exposure to PilVax, irrespective of the presence of the peptide, resulted in a significant reduction in S. aureus load in both the intestine and nasal mucosa (both P < 0.05) when compared with unvaccinated control mice. The mechanism(s) of protection are unclear, but merit further investigation to determine whether PilVax is a suitable platform for delivery of vaccine candidate antigens to the mucosa. RESULTS Characterization of L. lactis PilM1 vaccine strainsAttempts were made to express S. aureus FnBP peptides D1, D3 and FnBP10 (Supplementary table 1) within the bE-bF, b3-b4 and b9-b10 loop regions of L. lactis 370 PilVax elicits mucosal immunity to S. aureus F Clow et al.
The DNA damage response (DDR) comprises a range of mechanisms that ensure the integrity of the genome. As part of the DDR and in response to specific DNA damage, the master kinases ATM, ATR and DNA-PK are activated in a coordinated fashion inducing cell cycle arrest and repair of the damage before resuming DNA replication. Alternatively they may induce apoptosis if the DNA damage is incompatible with cell viability. Due to their genetic instability, tumour cells may be more reliant on the DNA damage response and so inhibiting specific DDR components may lead to antitumour activity while minimizing toxicity in normal tissue. ATR (Ataxia telangectasia and Rad 3 mutated) is recruited at specific sites of DNA damage, namely ssDNA (single stranded DNA) coated with RPA (Replication Protein A). ATR activates downstream substrates that regulate replication fork progression, cell cycle checkpoint, and DNA repair proteins. Here we report the discovery of AZ20, a novel potent and selective inhibitor of ATR kinase identified by screening a subset of the AZ compound collection against the target enzyme. In vitro, AZ20 decreases pChk1 Ser345, pChk1 Ser317 and pChk1 Ser296 levels in a concentration-dependent manner. Prolonged exposure with AZ20 increases γH2AX pan-nuclear staining, indicative of replication stress. This is associated with S-phase arrest and increase in phospho-histone H3. AZ20 induces growth inhibition and cell death in vitro and its profile of activity is distinct from other cytotoxic agents such as platinum derivatives, permetrexed or docetaxel. The cytotoxic effect of AZ20 can be increased in combination with the selective ATM inhibitor KU-60019. AZ20 induces significant tumour growth inhibition in vivo at well tolerated doses. This is associated with a persistent elevation of γH2AX pan-nuclear staining in xenograft tissue, but a transient increase in mouse bone marrow at therapeutic doses, suggesting a favourable therapeutic index. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1823. doi:1538-7445.AM2012-1823
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