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

Mosquito Larvicidal Potential of Gossypium hirsutum (Bt cotton) Leaves Extracts against Aedes aegypti and Anopheles stephensi larvae.

Background: We aimed to extract the ingredients from leaves of Gossypium hirsutum (Bt cotton) using different solvents and evaluate for potential use to control different larval stages of mosquito species, Aedes aegypti and Anopheles stephensi. Methods: Qualitative and quantitative estimation of ingredients from Go. hirsutum (Bt) plant extract was carried out and their inhibitory action against mosquito larvae was determined using mosquito larvicidal assay. Results: LC50  values of water, ethanol, ethyl acetate and hexane extracts for Ae. aegypti were 211.73±21.49,241.64±19.92, 358.07±32.43, 401.03±36.19 and 232.56±26.00, 298.54±21.78, 366.50±30.59, 387.19±31.82 for 4th instar of An. stephensi, respectively. The water extract displayed lowest LC50 value followed by ethanol, ethyl acetate and hexane. Owing to the comparatively better activity of water extract, its efficacy was further evaluated for mos- quito larvicidal activity, which exhibited LC50  values of 133.95±12.79, 167.65±11.34 against 2nd  and 3rd  instars of Ae. aegypti and 145.48±11.76, 188.10±12.92 against 2nd and 3rd instars of An. stephensi, respectively. Crude protein from the water extract was precipitated using acetone and tested against 2nd, 3rd and 4th instars of Ae. aegypti and An. stephensi. It revealed further decrease in LC50 values as 105.72±25.84, 138.23±23.18, 126.19±25.65, 134.04±04 and 137.88±17.59, 154.25±16.98 for 2nd, 3rd and 4th instars of Ae. aegypti and An. stephensi, respectively. Conclusion: Leaves extracts of Go. hirsutum (Bt) is potential mosquito larvicide and can be used as a potent alter- native to chemical insecticides in integrated pest management

Heparan Sulfate Binding Cationic Peptides Restrict SARS-CoV-2 Entry

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. While the world is striving for a treatment modality against SARS-CoV-2, our understanding about the virus entry mechanisms may help to design entry inhibitors, which may help to limit the virus spreading. Owing to the importance of cellular ACE2 and heparan sulfate in SARS-CoV-2 entry, we aimed to evaluate the efficacy of cationic G1 and G2 peptides in virus entry inhibition. In silico binding affinity studies revealed possible binding sites of G1 and G2 peptides on HS and ACE2, which are required for the spike–HS and spike–ACE2 interactions. Prophylactic treatment of G1 and G2 peptide was also proved to decrease the cell surface HS, an essential virus entry receptor. With these two mechanisms we confirm the possible use of cationic peptides to inhibit the entry of SARS-CoV-2

Potentiation of <i>Bacillus thuringiensis</i> by using some natural products: Novel preparations against dengue vector <i>Aedes aegypti</i> larvae

229-233Dengue fever is the fastest emerging arboviral infection causing millions of deaths all over the world. The eradication of vector Aedes aegypti, is an effective method of dengue control. Although various vector control agents like chemical pesticides are available, Bacillus thuringiensis (Bt) is of major choice as a biocontrol agent due to its ecofriendly nature. In the present investigation, curcumin, plumbagin, camphor, rutin, quercetin, karanjin, and pongamal were used as Bt SV2 potentiating agents. It was observed that curcumin and rutin had very high LC50 values for fourth instar larvae of Ae. aegypti that indicates lower activity. Karanjincaused significantly high mortality at comparatively low dose (LC50 - 44.59 ppm). At the same timepongamal, plumbagin, and camphorcaused significant mortalityat low doses of LC50 61.18, 59.23, and 71.59 ppm,respectively

Potentiation of Bacillus thuringiensis by using some natural products: Novel preparations against dengue vector Aedes aegypti larvae

Dengue fever is the fastest emerging arboviral infection causing millions of deaths all over the world. The eradication of vector Aedes aegypti, is an effective method of dengue control. Although various vector control agents like chemical pesticides are available, Bacillus thuringiensis (Bt) is of major choice as a biocontrol agent due to its ecofriendly nature. In the present investigation, curcumin, plumbagin, camphor, rutin, quercetin, karanjin, and pongamal were used as Bt SV2 potentiating agents. It was observed that curcumin and rutin had very high LC50 values for fourth instar larvae of Ae. aegypti that indicates lower activity. Karanjincaused significantly high mortality at comparatively low dose (LC50 - 44.59 ppm). At the same timepongamal, plumbagin, and camphorcaused significant mortalityat low doses of LC50 61.18, 59.23, and 71.59 ppm, respectively

Mercapto-pyrimidines are reversible covalent inhibitors of the papain-like protease (PLpro) and inhibit SARS-CoV-2 (SCoV-2) replication

The papain-like protease (PLpro) plays a critical role in SARS-CoV-2 (SCoV-2) pathogenesis and is essential for viral replication and for allowing the virus to evade the host immune response. Inhibitors of PLpro have great therapeutic potential, however, developing them has been challenging due to PLpro's restricted substrate binding pocket. In this report, we screened a 115 000-compound library for PLpro inhibitors and identified a new pharmacophore, based on a mercapto-pyrimidine fragment that is a reversible covalent inhibitor (RCI) of PLpro and inhibits viral replication in cells. Compound 5 had an IC50 of 5.1 μM for PLpro inhibition and hit optimization yielded a derivative with increased potency (IC50 0.85 μM, 6-fold higher). Activity based profiling of compound 5 demonstrated that it reacts with PLpro cysteines. We show here that compound 5 represents a new class of RCIs, which undergo an addition elimination reaction with cysteines in their target proteins. We further show that their reversibility is catalyzed by exogenous thiols and is dependent on the size of the incoming thiol. In contrast, traditional RCIs are all based upon the Michael addition reaction mechanism and their reversibility is base-catalyzed. We identify a new class of RCIs that introduces a more reactive warhead with a pronounced selectivity profile based on thiol ligand size. This could allow the expansion of RCI modality use towards a larger group of proteins important for human disease

Mild SARS-CoV-2 infection results in long-lasting microbiota instability

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2-positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls. These results were confirmed and extended in the K18-humanized angiotensin-converting enzyme 2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the USA), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology.IMPORTANCETaken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2, it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease