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

Gene Set Enrichment Analysis Using Single Nucleotide Polymorphisms to Identify Genes Associated with Residual Feed Intake in Cattle

Feed comprises 66% and 77% of the total cost of beef cattle calf and yearling finishing systems, respectively. Heritabilities for feed intake and feed efficiency (FE, estimated as residual feed intake, or RFI) have ranged from 0.08 to 0.46 in previous studies, highlighting the potential for genetic selection to bring about significant gains in feed efficiency and profitability within the beef industry. The objective of this study was to identify gene pathways significant for FE (as measured by RFI) through the use of Gene set enrichment analysis-single nucleotide polymorphism (GSEA-SNP) using single nucleotide polymorphisms (SNPs) as proxies for bovine genes. A population of 847 Hereford cattle (181 purebreds and 666 Hereford cross animals) consisting of 23 females and 824 males ranging in age from 210 to 496 d from a single ranch were evaluated for a period ranging from 70 to 140 days on feed (DOF). Only 31 animals were fed over 72 days. Average daily gain (ADG), dry matter intake (DMI), initial weight (IW), mid-test metabolic weight (MMWT), and DOF were recorded across the feeding period for each individual. Covariates for the genome-wide association study (GWAS) were age, % Hereford, and a series of 6 contemporary groups based on harvest date. GWAS was followed by GSEA-SNP of SNP data with Bos taurus gene sets sourced from GO, KEGG, Panther, Reactome, and Metacyc. A total of 20,692 bovine genes were mapped within gene sets, and proxy SNPs were mapped to genes located within 20 kilobase pairs. The null distribution of the GSEA-SNP test statistic was approximated using 10,000 permutations. A majority of genotypes were obtained from the Illumina bovine HD BeadChip, while the remainder were obtained using the Illumina bovine 50K BeadChip and imputed to 778,000 SNPs using Beagle. The Metacyc pathway ‘metabolism of proteins’ with 257 genes tended towards significance for RFI with a normalized enrichment score (NES) of 2.917. There were a total of 108 leading edge genes in the pathway. The top 10 of the 108 genes were: DNAJB11, RPL24, RPS3, PROS1, EIF4A2, ST6GAL1, RPS14, TBCE, EIF3I, and CCL2. Heritability for RFI was estimated to be 0.30. These results suggest that genetic selection for RFI has potential to improve the efficiency and, therefore, profitability of beef cattle production

Data from: Impact of confinement housing on study end-points in the calf model of cryptosporidiosis

Background: Diarrhea is the second leading cause of death in children < 5 years globally and the parasite genus Cryptosporidium is a leading cause of that diarrhea. The global disease burden attributable to cryptosporidiosis is substantial and the only approved chemotherapeutic, nitazoxanide, has poor efficacy in HIV positive children. Chemotherapeutic development is dependent on the calf model of cryptosporidiosis, which is the best approximation of human disease. However, the model is not consistently applied across research studies. Data collection commonly occurs using two different methods: Complete Fecal Collection (CFC), which requires use of confinement housing, and Interval Collection (IC), which permits use of box stalls. CFC mimics human challenge model methodology but it is unknown if confinement housing impacts study end-points and if data gathered via this method is suitable for generalization to human populations. Methods: Using a modified crossover study design we compared CFC and IC and evaluated the impact of housing on study end-points. At birth, calves were randomly assigned to confinement (n = 14) or box stall housing (n = 9), or were challenged with 5 x 107 C. parvum oocysts, and followed for 10 days. Study end-points included fecal oocyst shedding, severity of diarrhea, degree of dehydration, and plasma cortisol. Findings: Calves in confinement had no significant differences in mean log oocysts enumerated per gram of fecal dry matter between CFC and IC samples (P = 0.6), nor were there diurnal variations in oocyst shedding (P = 0.1). Confinement housed calves shed significantly more oocysts (P = 0.05), had higher plasma cortisol (P = 0.001), and required more supportive care (P = 0.0009) than calves in box stalls. Conclusion: Housing method confounds study end-points in the calf model of cryptosporidiosis. Due to increased stress data collected from calves in confinement housing may not accurately estimate the efficacy of chemotherapeutics targeting C. parvum

Impact of confinement housing on study end-points in the calf model of cryptosporidiosis

<div><p>Background</p><p>Diarrhea is the second leading cause of death in children < 5 years globally and the parasite genus <i>Cryptosporidium</i> is a leading cause of that diarrhea. The global disease burden attributable to cryptosporidiosis is substantial and the only approved chemotherapeutic, nitazoxanide, has poor efficacy in HIV positive children. Chemotherapeutic development is dependent on the calf model of cryptosporidiosis, which is the best approximation of human disease. However, the model is not consistently applied across research studies. Data collection commonly occurs using two different methods: Complete Fecal Collection (CFC), which requires use of confinement housing, and Interval Collection (IC), which permits use of box stalls. CFC mimics human challenge model methodology but it is unknown if confinement housing impacts study end-points and if data gathered via this method is suitable for generalization to human populations.</p><p>Methods</p><p>Using a modified crossover study design we compared CFC and IC and evaluated the impact of housing on study end-points. At birth, calves were randomly assigned to confinement (n = 14) or box stall housing (n = 9), or were challenged with 5 x 10⁷ <i>C</i>. <i>parvum</i> oocysts, and followed for 10 days. Study end-points included fecal oocyst shedding, severity of diarrhea, degree of dehydration, and plasma cortisol.</p><p>Findings</p><p>Calves in confinement had no significant differences in mean log oocysts enumerated per gram of fecal dry matter between CFC and IC samples (<i>P</i> = 0.6), nor were there diurnal variations in oocyst shedding (<i>P</i> = 0.1). Confinement housed calves shed significantly more oocysts (<i>P</i> = 0.05), had higher plasma cortisol (<i>P</i> = 0.001), and required more supportive care (<i>P</i> = 0.0009) than calves in box stalls.</p><p>Conclusion</p><p>Housing method confounds study end-points in the calf model of cryptosporidiosis. Due to increased stress data collected from calves in confinement housing may not accurately estimate the efficacy of chemotherapeutics targeting <i>C</i>. <i>parvum</i>.</p></div

Calf Clinical Data

Sheet S1: Fecal oocyst shedding and severity of diarrhea for infected calves, including data on housing system, sample type, and sample time for days 0-10 post-infection. Sheet S2: Clinical outcomes for days 0-10 post-infection. Sheet S3: Calf body weights and calculation of average daily weight gain. Sheet S4: Frequency and type of morbidities and treatments for each calf. Sheet S5: Data Dictionar

Descriptive summary of study end-points for calves in confinement housing (n = 12) and box stalls (n = 9) from days 0 to 10 post-infection.

<p>Descriptive summary of study end-points for calves in confinement housing (n = 12) and box stalls (n = 9) from days 0 to 10 post-infection.</p

Descriptive summary of daily and stool and urine production for calves (n = 12) in confinement housing from days 0 to 10 post-infection.

<p>Descriptive summary of daily and stool and urine production for calves (n = 12) in confinement housing from days 0 to 10 post-infection.</p

Descriptive summary of supportive care administered to calves in confinement housing (n = 12) and box stalls (n = 9).

<p>Descriptive summary of supportive care administered to calves in confinement housing (n = 12) and box stalls (n = 9).</p

Comparison of plasma cortisol levels in confinement housing (CH) (n = 10) and box stall (BXS) calves (n = 5) experimentally infected with <i>C</i>. <i>parvum</i>.

<p>* P < 0.05.</p

Comparison (mean ± SE) of log oocysts enumerated per gram of fecal dry matter in confinement housing (CH) (n = 12) and box stall (BXS) (n = 9) calves experimentally infected with <i>C</i>. <i>parvum</i>.

<p>* P < 0.05. ** P < 0.006. *** P = 0.0001.</p