Insights into the Complex Formed by Matrix Metalloproteinase-2 and Alloxan Inhibitors: Molecular Dynamics Simulations and Free Energy Calculations

Matrix metalloproteinases (MMP) are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. Hence, the development of potent and selective inhibitors targeting these enzymes continues to be eagerly sought. In this paper, a number of alloxan-based compounds, initially conceived to bias other therapeutically relevant enzymes, were rationally modified and successfully repurposed to inhibit MMP-2 (also named gelatinase A) in the nanomolar range. Importantly, the alloxan core makes its debut as zinc binding group since it ensures a stable tetrahedral coordination of the catalytic zinc ion in concert with the three histidines of the HExxHxxGxxH metzincin signature motif, further stabilized by a hydrogen bond with the glutamate residue belonging to the same motif. The molecular decoration of the alloxan core with a biphenyl privileged structure allowed to sample the deep S1′ specificity pocket of MMP-2 and to relate the high affinity towards this enzyme with the chance of forming a hydrogen bond network with the backbone of Leu116 and Asn147 and the side chains of Tyr144, Thr145 and Arg149 at the bottom of the pocket. The effect of even slight structural changes in determining the interaction at the S1′ subsite of MMP-2 as well as the nature and strength of the binding is elucidated via molecular dynamics simulations and free energy calculations. Among the herein presented compounds, the highest affinity (pIC50 = 7.06) is found for BAM, a compound exhibiting also selectivity (>20) towards MMP-2, as compared to MMP-9, the other member of the gelatinases

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Comparative analysis of hemagglutination inhibition titers generated using temporally matched serum and plasma samples.

Influenza-specific hemaggluitination inhibition (HAI) antibody titer, an indicator of immunity to influenza, is often used to measure exposure to influenza in surveillance and immunogenicity studies. Traditionally, serum has been the specimen of choice for HAI assays, but a desire to reduce the amount of blood collected during studies and the availability of plasma in archived sample collections warrant the evaluation of plasma for HAI titer. Therefore, the relationship between serum and plasma HAI titer values is of great interest. Here, we compare HAI titers determined on temporally matched serum and plasma (citrated and heparinized) using influenza A and B viruses. Bland-Altman plots, McNemar's test, and geometric coefficient of variation were used respectively for evaluating agreement, correlation and variability in the serum-plasma titer results. We observed a high degree of agreement (80.5%-98.8%) and correlation (r = 0.796-0.964) in the serum and matched plasma titer values although plasma titers were generally lower than corresponding serum titers. Calculated seropositive (HAI ≥40) rates were higher using serum titers than with plasma titers, but seroconversion rates were unaffected by sample type. Stronger agreement and decreased variability in titers were seen between serum and citrated plasma than between serum and heparinized plasma. Overall, these data suggest that serum or plasma can be used in serodiagnostic HAI assays, but seropositive rates may be underestimated using plasma HAI titers. The type of anticoagulant present in plasma may affect HAI titer values and warrants further investigation

Nasopharyngeal Carriage and Transmission of <i>Streptococcus pneumoniae</i> in American Indian Households after a Decade of Pneumococcal Conjugate Vaccine Use

<div><p>Background</p><p>Young children played a major role in pneumococcal nasopharyngeal carriage, acquisition, and transmission in the era before pneumococcal conjugate vaccine (PCV) use. Few studies document pneumococcal household dynamics in the routine-PCV7 era.</p><p>Methods</p><p>We investigated age-specific acquisition, household introduction, carriage clearance, and intra-household transmission in a prospective, longitudinal, observational cohort study of pneumococcal nasopharyngeal carriage in 300 American Indian households comprising 1,072 participants between March 2006 and March 2008.</p><p>Results</p><p>Pneumococcal acquisition rates were 2–6 times higher in children than adults. More household introductions of new pneumococcal strains were attributable to children <9 years than adults ≥17 years (p<0.001), and older children (2–8 years) than younger children (<2 years) (p<0.008). Compared to children <2 years, carriage clearance was more rapid in older children (2–4 years, HR_clearance 1.53 [95% CI: 1.22, 1.91]; 5–8 years, HR_clearance 1.71 [1.36, 2.15]) and adults (HR_clearance 1.75 [1.16, 2.64]). Exposure to serotype-specific carriage in older children (2–8 years) most consistently increased the odds of subsequently acquiring that serotype for other household members.</p><p>Conclusions</p><p>In this community with a high burden of pneumococcal colonization and disease and routine PCV7 use, children (particularly older children 2–8 years) drive intra-household pneumococcal transmission: first, by acquiring, introducing, and harboring pneumococcus within the household, and then by transmitting acquired serotypes more efficiently than household members of other ages.</p></div