A Phase I, Open-Label Trial, Evaluating the Safety and Immunogenicity of Candidate Tuberculosis Vaccines AERAS-402 and MVA85A, Administered by Prime-Boost Regime in BCG-Vaccinated Healthy Adults.

BACKGROUND: MVA85A and AERAS-402 are two clinically advanced viral vectored TB vaccine candidates expressing Mycobacterium tuberculosis antigens designed to boost BCG-induced immunity. Clinical trials with candidate malaria vaccines have demonstrated that adenoviral vector based priming immunisation, followed by MVA vector boost, induced high levels of immunity. We present the safety and immunogenicity results of the first clinical trial to evaluate this immunisation strategy in TB. METHODS: In this phase 1, open-label trial, 40 healthy previously BCG-vaccinated participants were enrolled into three treatment groups and vaccinated with 1 or 2 doses of AERAS-402 followed by MVA85A; or 3 doses of AERAS-402. RESULTS: Most related adverse events (AEs) were mild and there were no vaccine related serious AEs. Boosting AERAS-402 with MVA85A significantly increased Ag85A-specific T-cell responses from day of vaccination. Two priming doses of AERAS-402 followed by MVA85A boost, resulted in a significantly higher AUC post-peak Ag85A response compared to three doses of AERAS-402 and historical data with MVA85A vaccination alone. The frequency of CD8+ T-cells producing IFN-γ, TNF-α and IL-2 was highest in the group receiving two priming doses of AERAS-402 followed by MVA85A. CONCLUSIONS: Vaccination with AERAS-402 followed by MVA85A was safe and increased the durability of antigen specific T-cell responses and the frequency and polyfunctionality of CD8+ T-cells, which may be important in protection against TB. Further clinical trials with adenoviral prime-MVA85A boost regimens are merited to optimise vaccination intervals, dose and route of immunisation and to evaluate this strategy in the target population in TB high burden countries. TRIAL REGISTRATION: ClinicalTrials.gov NCT01683773.

Ad35.CS.01-RTS,S/AS01 Heterologous Prime Boost Vaccine Efficacy against Sporozoite Challenge in Healthy Malaria-Naïve Adults.

METHODS: In an observer blind, phase 2 trial, 55 adults were randomized to receive one dose of Ad35.CS.01 vaccine followed by two doses of RTS,S/AS01 (ARR-group) or three doses of RTS,S/AS01 (RRR-group) at months 0, 1, 2 followed by controlled human malaria infection. RESULTS: ARR and RRR vaccine regimens were well tolerated. Efficacy of ARR and RRR groups after controlled human malaria infection was 44% (95% confidence interval 21%-60%) and 52% (25%-70%), respectively. The RRR-group had greater anti-CS specific IgG titers than did the ARR-group. There were higher numbers of CS-specific CD4 T-cells expressing > 2 cytokine/activation markers and more ex vivo IFN-γ enzyme-linked immunospots in the ARR-group than the RRR-group. Protected subjects had higher CS-specific IgG titers than non-protected subjects (geometric mean titer, 120.8 vs 51.8 EU/ml, respectively; P = .001). CONCLUSIONS: An increase in vaccine efficacy of ARR-group over RRR-group was not achieved. Future strategies to improve upon RTS,S-induced protection may need to utilize alternative highly immunogenic prime-boost regimens and/or additional target antigens. TRIAL REGISTRATION: ClinicalTrials.gov NCT01366534.

A phase 1b randomized, controlled, double-blinded dosage-escalation trial to evaluate the safety, reactogenicity and immunogenicity of an adenovirus type 35 based circumsporozoite malaria vaccine in Burkinabe healthy adults 18 to 45 years of age.

BACKGROUND: Ad35.CS.01 is a pre-erythrocytic malaria candidate vaccine. It is a codon optimized nucleotide sequence representing the P. falciparum circumsporozoite (CS) surface antigen inserted in a replication deficient Adenovirus 35 backbone. A Phase 1a trial has been conducted in the USA in naïve adults and showed that the vaccine was safe. The aim of this study is to assess the safety and immunogenicity of ascending dosages in sub Saharan Africa. METHODS: A double blind, randomized, controlled, dose escalation, phase Ib trial was conducted in a rural area of Balonghin, the Saponé health district (Burkina Faso). Forty-eight healthy adults aged 18-45 years were randomized into 4 cohorts of 12 to receive three vaccine doses (day 0, 28 and 84) of 10(9), 10(10), 5X10(10), 10(11) vp of Ad35.CS.01 or normal saline by intra muscular injection. Subjects were monitored carefully during the 14 days following each vaccination for non serious adverse events. Severe and serious adverse events were collected throughout the participant study duration (12 months from the first vaccination). Humoral and cellular immune responses were measured on study days 0, 28, 56, 84, 112 and 140. RESULTS: Of the forty-eight subjects enrolled, forty-four (91.7%) received all three scheduled vaccine doses. Local reactions, all of mild severity, occurred in thirteen (27.1%) subjects. Severe (grade 3) laboratory abnormalities occurred in five (10.4%) subjects. One serious adverse event was reported and attributed to infection judged unrelated to vaccine. The vaccine induced both antibody titers and CD8 T cells producing IFNγ and TNFα with specificity to CS while eliciting modest neutralizing antibody responses against Ad35. CONCLUSION: Study vaccine Ad35.CS.01 at four different dose levels was well-tolerated and modestly immunogenic in this population. These results suggest that Ad35.CS.01 should be further investigated for preliminary efficacy in human challenge models and as part of heterologous prime-boost vaccination strategies. TRIAL REGISTRATION: ClinicalTrials.gov NCT01018459 http://clinicaltrials.gov/ct2/show/NCT01018459.

Randomized, placebo-controlled trial to assess the safety and immunogenicity of an adenovirus type 35-based circumsporozoite malaria vaccine in healthy adults.

Malaria results in over 650,000 deaths each year; thus, there is an urgent need for an effective vaccine. Pre-clinical studies and recently reported human trials suggest that pre-erythrocytic stage vaccines can provide protection against infection. A Phase 1, randomized, placebo-controlled, dose-escalation study was conducted with a vaccine composed of a replication-deficient adenovirus-35 backbone with P. falciparum circumsporozoite (CS) surface antigen (Ad35.CS.01). Healthy adult subjects received three doses of 10 (8), 10 (9), 10 (10), or 10 (11) vp/mL Ad35.CS.01 vaccine or saline placebo intramuscularly at 0, 1, and 6-mo intervals. Adverse events were assessed and anti-CS antibody responses were determined by ELISA. Seventy-two individuals were enrolled, with age, gender, and ethnicity similar across each study arm. While the vaccine was generally well tolerated, adverse events were more frequent in the highest dose groups (10 (10) and 10 (11) vp/mL). More robust humoral responses were also noted at the highest doses, with 73% developing a positive ELISA response after the three dose series of 10 (11) vp/mL. The Ad35.CS.01 vaccine was most immunogenic at the highest dosages (10 (10) and 10 (11) vp/mL). Reactogenicity findings were more common after the 10 (11) vp/mL dose, although most were mild or moderate in nature and resolved without therapy.

An unusual DNA binding compound, S23906, induces mitotic catastrophe in cultured human cells.

The biochemical pathways that lead cells to mitotic catastrophe are not well understood. To identify these pathways, we have taken an approach of treating cells with a novel genotoxic compound and characterizing whether cells enter mitotic catastrophe or not. S23906 is a novel acronycine derivative that forms adducts with the N2 residue of guanine in the minor groove of the DNA helix and destabilizes base pairing to cause helix opening. We observed, in HeLa and HT-29 cells, that S23906 induced gamma-H2AX and activated checkpoint kinase 1, as did bleomycin, camptothecin, and cisplatin, when tested under equi-toxic conditions. S23906 also induced cyclin E1 protein, although this activity was not required for cytotoxicity because knock down of cyclin E1 by RNA interference did not affect the number of dead cells after treatment. Cyclin B1 levels first decreased and then increased after treatment with S23906. Cyclin B1 was associated with Cdk1 kinase activity, which correlated with an increase in the number of mitotic cells. By 32 h after treatment, at least 20% of the cells entered mitotic catastrophe as determined by microscopy. Suppression of the DNA checkpoint response by co-treatment with caffeine increased the number of cells in mitosis. These results suggest that mitotic catastrophe is one of the cellular responses to S23906 and that mitotic catastrophe may be a common cellular response to many different types of DNA damage.

The tumor suppressor hSNF5/INI1 modulates cell growth and actin cytoskeleton organization.

hSNF5/INI1, which encodes a component of the ATP-dependent chromatin remodeling hSWI-SNF complex, is a tumor suppressor gene mutated in malignant rhabdoid tumors. We have developed a tetracycline-based hSNF5/INI1-inducible system in a hSNF5/INI1-deficient malignant rhabdoid tumor cell line and studied time course variation of 22,000 genes/expressed sequence tags upon hSNF5/INI1 induction. A total of 482 responsive genes were identified and further clustered into 9 groups of coregulated genes. Among genes with early and strong inductions, the use of a fusion protein with the hormone-binding domain of the estrogen receptor enabled the identification of a subset of direct targets regulated independently of de novo protein synthesis. We show that the G(1) arrest induced by hSNF5/INI1 is reversible and associated with the down-regulation of components of the DNA replication complex. We also identify an unsuspected role of hSNF5/INI1 in cytoskeleton organization. Indeed, induction of hSNF5/INI1 induces dramatic modifications of the cell shape including complete disruption of the actin stress fiber network and disappearance of focal adhesions associated with up-regulation of genes involved in the organization of the actin cytoskeleton. We document a strong decrease of Rho activity upon hSNF5/INI1 expression, suggesting that the regulation of this activity constitutes a crucial step of the hSNF5/INI1-induced reorganization of the actin network. This study identifies hSNF5/INI1 target genes and provides evidence that hSNF5/INI1 may modulate the cell cycle control and cytoskeleton organization through the regulation of the retinoblastoma protein-E2F and Rho pathways.

A key role of the hSNF5/INI1 tumour suppressor in the control of the G1-S transition of the cell cycle.

The hSNF5/INI1 gene encodes a member of the SWI/SNF chromatin remodelling complexes. It was recently identified as a tumour suppressor gene mutated in sporadic and hereditary Malignant Rhabdoid Tumours (MRT). However, the role of hSNF5/INI1 loss-of-function in tumour development is still unknown. Here, we show that the ectopic expression of wild-type hSNF5/INI1, but not that of truncated versions, leads to a cell cycle arrest by inhibiting the entry into S phase of MRT cells. This G1 arrest is associated with down-regulation of a subset of E2F targets including cyclin A, E2F1 and CDC6. This arrest can be reverted by coexpression of cyclin D1, cyclin E or viral E1A, whereas it cannot be counteracted by pRB-binding deficient E1A mutants. Moreover, hSNF5/INI1 is not able to arrest cells lacking a functional pRB. These observations suggest that the hSNF5/INI1-induced G1 arrest is dependent upon the presence of a functional pRB. However, the observation that a constitutively active pRB can efficiently arrest MRT cells indicates that hSNF5/INI1, at the difference of the ATPase subunits of the SWI/SNF complex, is dispensable for pRB function. Altogether, these data show that hSNF5/INI1 is a potent regulator of the entry into S phase, an effect that may account for its tumour suppressor role.