A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis : First-in-human trial of ChAd63-KH

BACKGROUND: Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells. METHODS: We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. FINDINGS: ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. CONCLUSION: The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. TRIAL REGISTRATION: This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359)

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Genetic variants influencing biomarkers of nutrition are not associated with cognitive capability in middle-aged and older adults

Several investigations have observed positive associations between good nutritional status, as indicated by micronutrients, and cognitive measures; however, these associations may not be causal. Genetic polymorphisms that affect nutritional biomarkers may be useful for providing evidence for associations between micronutrients and cognitive measures. As part of the Healthy Ageing across the Life Course (HALCyon) program, men and women aged between 44 and 90 y from 6 UK cohorts were genotyped for polymorphisms associated with circulating concentrations of iron [rs4820268 transmembrane protease, serine 6 (TMPRSS6) and rs1800562 hemochromatosis (HFE)], vitamin B-12 [(rs492602 fucosyltransferase 2 (FUT2)], vitamin D ([rs2282679 group-specific component (GC)] and β-carotene ([rs6564851 beta-carotene 15,15'-monooxygenase 1 (BCMO1)]. Meta-analysis was used to pool within-study effects of the associations between these polymorphisms and the following measures of cognitive capability: word recall, phonemic fluency, semantic fluency, and search speed. Among the several statistical tests conducted, we found little evidence for associations. We found the minor allele of rs1800562 was associated with poorer word recall scores [pooled β on Z-score for carriers vs. noncarriers: -0.05 (95% CI: -0.09, -0.004); P = 0.03, n = 14,105] and poorer word recall scores for the vitamin D-raising allele of rs2282679 [pooled β per T allele: -0.03 (95% CI: -0.05, -0.003); P = 0.03, n = 16,527]. However, there was no evidence for other associations. Our findings provide little evidence to support associations between these genotypes and cognitive capability in older adults. Further investigations are required to elucidate whether the previous positive associations from observational studies between circulating measures of these micronutrients and cognitive performance are due to confounding and reverse causality

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Vaccination with the surface proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans induces antibodies but fails to provide protection against Buruli ulcer

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters.; To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a TH1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model.; Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci

Immunogenicity of recombinant protein/EM048 formulation in comparison to commercially available adjuvants for mice.

<p>Groups of five BALB/c mice were immunized three times in three week intervals with 20 μg of rMUL2232 (A) or rMUL3720 (B) in PBS or formulated with Alum, Sigma Adjuvant or EM048. Serum three weeks after every immunization (I1, I2 and I3) was analysed in ELISA on the respective recombinant protein. Depicted are individual endpoint IgG titers as determined in one single ELISA, the mean (line) ± standard deviation. Values that are zero are not depicted but were included for statistical analysis. Overall statistical significance was calculated by Kruskal-Wallis test per immunization time point (A: P_I1 = 0.0018, P_I2 = 0.0372 and P_I3 = ns.; B: P_I1 = 0.0095, P_I2 = 0.0011, P_I3 = 0.0041.). Individual statistical differences between groups were assessed by the Dunn procedure and are depicted if detected (* <i>p</i> ≤ 0.05; ** <i>p</i> ≤ 0.01).</p

Evaluation of the protective potential of immunization with rMUL3720/EM048 formulation in a <i>M</i>. <i>ulcerans</i> infection mouse model.

<p>Groups of eight BALB/c mice were immunized twice with 20 μg of rMUL3720/EM048 or PBS alone as infection control. Three weeks after the last immunization mice were challenged with a high dose or a low dose of <i>M</i>. <i>ulcerans</i> (inoculum) into the left hind foot pad. Infection was followed by measuring foot pad thickness with a caliper (A) until mice were euthanized at day 63 after infection (high dose, H1) or at day 87 after infection (low dose, H2). Depicted is the mean foot pad thickness (diamond/circle) ± standard deviation of the differently immunized groups. (B) Bacterial load in infected foot pads was determined by qPCR for six mice per group. Depicted are individual measurements as genome copies per foot pad, the mean (line) ± standard deviation.</p

Cross reactivity of the immune sera with <i>M</i>. <i>ulcerans</i>.

<p>(A) Groups of five BALB/c mice (m1 –m5) were immunized three times in three week intervals with 20 μg of rMUL2232 formulated with Alum, Sigma Adjuvant or EM048. Serum three weeks after every immunization (I1, I2 and I3) was analysed by Western blotting on <i>M</i>. <i>ulcerans</i> lysate. Monoclonal anti-MUL_2232 antibody (mAb) served as positive control, pre-bleed (pb) serum or no primary antibody (nc) as negative controls. (B) Sera from three weeks after the third immunization with rMUL2232 and indicated adjuvant were used for indirect immunofluorescence staining on paraffin embedded <i>M</i>. <i>ulcerans</i> bacteria with an Alexa488 labelled secondary antibody. Pre-bleed serum did not stain the bacteria. Sera of immunized mice (a mix of sera from five individual mice per group) did reveal surface staining similar to the staining achieved with anti-MUL_2232 monoclonal antibody (mAb).</p

Evaluation of the booster effect of <i>M</i>. <i>ulcerans</i> infection on the pre-existing antibody response in rMUL3720/EM048 immunized and subsequently infected mice.

<p>(A1) Serum of six BALB/c mice immunized with rMUL2232/EM048 prior to infection (before infection) with <i>M</i>. <i>ulcerans</i> was compared to serum of the same animals after 42 days of infection (after infection) in ELISA on rMUL2232. Control immunized animals had received PBS/EM048 or only PBS prior to infection. Depicted are individual endpoint IgG titers as determined in one single ELISA, the group mean (line) ± standard deviation. Statistical significance was calculated by the Student’s t- test (** <i>p</i> ≤ 0.01). (A2) Serum of eight BALB/c mice immunized with rMUL3720/EM048 prior to infection with <i>M</i>. <i>ulcerans</i> was compared to serum of the same animals 63 days after infection with <i>M</i>. <i>ulcerans</i> bacteria in ELISA on rMUL3720. Control immunized animals (control) had only received PBS prior to infection. Depicted are individual endpoint IgG titers as determined in one single ELISA, the mean (line) ± standard deviation. Statistical significance was calculated by the Student’s t- test (**** <i>p</i> ≤ 0.0001). (B) Serum of six BALB/c mice (m1 –m6) immunized with rMUL3720/EM048 prior to infection with <i>M</i>. <i>ulcerans</i> (a) was compared to serum of the same animals after 42 days of infection (b) by Western blotting on <i>M</i>. <i>ulcerans</i> whole cell lysate. Monoclonal anti-MUL_3720 antibody (mAb) served as positive control, pre-bleed (pb) serum or no primary antibody (nc) as negative controls.</p