Safety, tolerability, and immunogenicity of influenza vaccination with a high-density microarray patch: Results from a randomized, controlled phase I clinical trial.

BACKGROUND: The Vaxxas high-density microarray patch (HD-MAP) consists of a high density of microprojections coated with vaccine for delivery into the skin. Microarray patches (MAPs) offer the possibility of improved vaccine thermostability as well as the potential to be safer, more acceptable, easier to use, and more cost-effective for the administration of vaccines than injection by needle and syringe (N&S). Here, we report a phase I trial using the Vaxxas HD-MAP to deliver a monovalent influenza vaccine that was to the best of our knowledge the first clinical trial to evaluate the safety, tolerability, and immunogenicity of lower doses of influenza vaccine delivered by MAPs. METHODS AND FINDINGS: HD-MAPs were coated with a monovalent, split inactivated influenza virus vaccine containing A/Singapore/GP1908/2015 H1N1 haemagglutinin (HA). Between February 2018 and March 2018, 60 healthy adults (age 18-35 years) in Melbourne, Australia were enrolled into part A of the study and vaccinated with either: HD-MAPs delivering 15 µg of A/Singapore/GP1908/2015 H1N1 HA antigen (A-Sing) to the volar forearm (FA); uncoated HD-MAPs; intramuscular (IM) injection of commercially available quadrivalent influenza vaccine (QIV) containing A/Singapore/GP1908/2015 H1N1 HA (15 µg/dose); or IM injection of H1N1 HA antigen (15 µg/dose). After 22 days' follow-up and assessment of the safety data, a further 150 healthy adults were enrolled and randomly assigned to 1 of 9 treatment groups. Participants (20 per group) were vaccinated with HD-MAPs delivering doses of 15, 10, 5, 2.5, or 0 µg of HA to the FA or 15 µg HA to the upper arm (UA), or IM injection of QIV. The primary objectives of the study were safety and tolerability. Secondary objectives were to assess the immunogenicity of the influenza vaccine delivered by HD-MAP. Primary and secondary objectives were assessed for up to 60 days post-vaccination. Clinical staff and participants were blind as to which HD-MAP treatment was administered and to administration of IM-QIV-15 or IM-A/Sing-15. All laboratory investigators were blind to treatment and participant allocation. Two further groups in part B (5 participants per group), not included in the main safety and immunological analysis, received HD-MAPs delivering 15 µg HA or uncoated HD-MAPs applied to the forearm. Biopsies were taken on days 1 and 4 for analysis of the cellular composition from the HD-MAP application sites. The vaccine coated onto HD-MAPs was antigenically stable when stored at 40°C for at least 12 months. HD-MAP vaccination was safe and well tolerated; any systemic or local adverse events (AEs) were mild or moderate. Observed systemic AEs were mostly headache or myalgia, and local AEs were application-site reactions, usually erythema. HD-MAP administration of 2.5 µg HA induced haemagglutination inhibition (HAI) and microneutralisation (MN) titres that were not significantly different to those induced by 15 µg HA injected IM (IM-QIV-15). HD-MAP delivery resulted in enhanced humoral responses compared with IM injection with higher HAI geometric mean titres (GMTs) at day 8 in the MAP-UA-15 (GMT 242.5, 95% CI 133.2-441.5), MAP-FA-15 (GMT 218.6, 95% CI 111.9-427.0), and MAP-FA-10 (GMT 437.1, 95% CI 254.3-751.3) groups compared with IM-QIV-15 (GMT 82.8, 95% CI 42.4-161.8), p = 0.02, p = 0.04, p < 0.001 for MAP-UA-15, MAP-FA-15, and MAP-FA-10, respectively. Higher titres were also observed at day 22 in the MAP-FA-10 (GMT 485.0, 95% CI 301.5-780.2, p = 0.001) and MAP-UA-15 (367.6, 95% CI 197.9-682.7, p = 0.02) groups compared with the IM-QIV-15 group (GMT 139.3, 95% CI 79.3-244.5). Results from a panel of exploratory immunoassays (antibody-dependent cellular cytotoxicity, CD4+ T-cell cytokine production, memory B cell (MBC) activation, and recognition of non-vaccine strains) indicated that, overall, Vaxxas HD-MAP delivery induced immune responses that were similar to, or higher than, those induced by IM injection of QIV. The small group sizes and use of a monovalent influenza vaccine were limitations of the study. CONCLUSIONS: Influenza vaccine coated onto the HD-MAP was stable stored at temperatures up to 40°C. Vaccination using the HD-MAP was safe and well tolerated and resulted in immune responses that were similar to or significantly enhanced compared with IM injection. Using the HD-MAP, a 2.5 µg dose (1/6 of the standard dose) induced HAI and MN titres similar to those induced by 15 µg HA injected IM. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR.org.au), trial ID 108 ACTRN12618000112268/U1111-1207-3550.

Surgeon volume and 30 day mortality for brain tumours in England.

BACKGROUND: There is evidence that surgeons who perform more operations have better outcomes. However, in patients with brain tumours, all of the evidence comes from the USA. METHODS: We examined all English patients with an intracranial neoplasm who had an intracranial resection in 2008-2010. We included surgeons who performed at least six operations over 3 years, and at least one operation in the first and last 6 months of the period. RESULTS: The analysis data set comprised 9194 operations, 163 consultant neurosurgeons and 30 centres. Individual surgeon volumes varied widely (7-272; median=46). 72% of operations were on the brain, and 30 day mortality was 3%. A doubling of surgeon load was associated with a 20% relative reduction in mortality. Thirty day mortality varied between centres (0·95-8·62%) but was not related to centre workload. CONCLUSIONS: Individual surgeon volumes correlated with patient 30 day mortality. Centres and surgeons in England are busier than surgeons and centres in the USA. There is no relationship between centre volume and 30 day mortality in England. Services in the UK appear to be adequately arranged at a centre level, but would benefit from further surgeon sub-specialisation.

Analysis of pre-treatment markers predictive of treatment benefit for the therapeutic cancer vaccine MVA-5T4 (TroVax).

Cancer vaccines such as MVA-5T4 (TroVax(®)) must induce an efficacious immune response to deliver therapeutic benefit. The identification of biomarkers that impact on the clinical and/or immunological efficacy of cancer vaccines is required in order to select patients who are most likely to benefit from this treatment modality. Here, we sought to identify a predictor of treatment benefit for renal cancer patients treated with MVA-5T4. Statistical modeling was undertaken using data from a phase III trial in which patients requiring first-line treatment for metastatic renal cell carcinoma were randomized 1:1 to receive MVA-5T4 or placebo alongside sunitinib, IL-2 or IFN-α. Numerous pre-treatment factors associated with inflammatory anemia (e.g., CRP, hemoglobin, hematocrit, IL-6, ferritin, platelets) demonstrated a significant relationship with tumor burden and patient survival. From these prognostic factors, the pre-treatment mean corpuscular hemoglobin concentration (MCHC) was found to be the best predictor of treatment benefit (P < 0.01) for MVA-5T4 treated patients and also correlated positively with tumor shrinkage (P < 0.001). Furthermore, MCHC levels showed a significant positive association with 5T4 antibody response (P = 0.01). The latter result was confirmed using an independent data set comprising phase II trials of MVA-5T4 in patients with colorectal, renal and prostate cancers. Retrospective analyses demonstrated that RCC patients who had very large tumor burdens and low MCHC levels received little or no benefit from treatment with MVA-5T4; however, patients with smaller tumor burdens and normal MCHC levels received substantial benefit from treatment with MVA-5T4.

MVA-5T4-induced immune responses are an early marker of efficacy in renal cancer patients.

Few immunotherapy compounds have demonstrated a direct link between the predicted mode of action of the product and benefit to the patient. Since cancer vaccines are thought to have a delayed therapeutic effect, identification of the active moiety may enable the development of an early marker of efficacy. Patients with renal cancer and requiring first-line treatment for metastatic disease were randomized 1:1 to receive MVA-5T4 (TroVax(®)) or placebo alongside Sunitinib, IL-2 or IFN-α in a multicentre phase III trial. Antibody responses were quantified following the 3rd and 4th vaccinations. A surrogate for 5T4 antibody response (the immune response surrogate; IRS) was constructed and then used in a survival analysis to evaluate treatment benefit. Seven hundred and thirty-three patients were randomized, and immune responses were assessed in 590 patients. A high 5T4 antibody response was associated with longer survival within the MVA-5T4-treated group. The IRS was constructed as a linear combination of pre-treatment 5T4 antibody levels, hemoglobin and hematocrit and was shown to be a significant predictor of treatment benefit in the phase III study. Importantly, the IRS was also associated with antibody response and survival in an independent dataset comprising renal, colorectal and prostate cancer patients treated with MVA-5T4 in phase I-II studies. The derivation of the IRS formed part of an exploratory, retrospective analysis; however, if confirmed in future studies, the results have important implications for the development and use of the MVA-5T4 vaccine and potentially for other similar vaccines.

Cellular responses at the application site of a high-density microarray patch delivering an influenza vaccine in a randomized, controlled phase I clinical trial.

Microarray patches (MAPs) have the potential to be a safer, more acceptable, easier to use and more cost-effective method for administration of vaccines when compared to the needle and syringe. Since MAPs deliver vaccine to the dermis and epidermis, a degree of local immune response at the site of application is expected. In a phase 1 clinical trial (ACTRN 12618000112268), the Vaxxas high-density MAP (HD-MAP) was used to deliver a monovalent, split inactivated influenza virus vaccine into the skin. HD-MAP immunisation led to significantly enhanced humoral responses on day 8, 22 and 61 compared with IM injection of a quadrivalent commercial seasonal influenza vaccine (Afluria Quadrivalent®). Here, the aim was to analyse cellular responses to HD-MAPs in the skin of trial subjects, using flow cytometry and immunohistochemistry. HD-MAPs were coated with a split inactivated influenza virus vaccine (A/Singapore/GP1908/2015 [H1N1]), to deliver 5 µg haemagglutinin (HA) per HD-MAP. Three HD-MAPs were applied to the volar forearm (FA) of five healthy volunteers (to achieve the required 15 µg HA dose), whilst five control subjects received three uncoated HD-MAPs (placebo). Local skin response was recorded for over 61 days and haemagglutination inhibition antibody titres (HAI) were assessed on days 1, 4, 8, 22, and 61. Skin biopsies were taken before (day 1), and three days after HD-MAP application (day 4) and analysed by flow-cytometry and immunohistochemistry to compare local immune subset infiltration. HD-MAP vaccination with 15 µg HA resulted in significant HAI antibody titres compared to the placebo group. Application of uncoated placebo HD-MAPs resulted in mild erythema and oedema in most subjects, that resolved by day 4 in 80% of subjects. Active, HA-coated HD-MAP application resulted in stronger erythema responses on day 4, which resolved between days 22-61. Overall, these erythema responses were accompanied by an influx of immune cells in all subjects. Increased cell infiltration of CD3+, CD4+, CD8+ T cells as well as myeloid CD11b+ CD11c+ and non-myeloid CD11b- dendritic cells were observed in all subjects, but more pronounced in active HD-MAP groups. In contrast, CD19+/CD20+ B cell counts remained unchanged. Key limitations include the use of an influenza vaccine, to which the subjects may have had previous exposure. Different results might have been obtained with HD-MAPs inducing a primary immune response. In conclusion, influenza vaccine administered to the forearm (FA) using the HD-MAP was well-tolerated and induced a mild to moderate skin response with lymphocytic infiltrate at the site of application.

Efficacy and Safety of CAP7.1 as Second-Line Treatment for Advanced Biliary Tract Cancers: Data from a Randomised Phase II Study.

CAP7.1 is a novel topoisomerase II inhibitor, converted to active etoposide via carboxylesterase 2 (CES2), with signals of efficacy in treatment-refractory solid tumours. In a Phase II trial, 27 patients with advanced biliary tract cancers (BTC) were randomised 1:1 to CAP7.1 plus best supportive care (BSC), or BSC alone, with crossover to CAP7.1 upon disease progression. The primary objective was disease control rate (DCR) following 28-day cycles of CAP7.1 (200/150 mg/m2; iv), or BSC until progression. Secondary objectives included progression-free survival (PFS), time-to-treatment failure (TTF), overall survival (OS) and safety. Fourteen patients received CAP7.1 and 13 BSC. DCR favoured CAP7.1 vs. BSC (50% vs. 20%; treatment difference: 30%, 95%CI -18.44, 69.22, full analysis set [FAS]), with disease progression in 40% vs. 70%, respectively. Significantly longer median PFS was achieved for CAP7.1 vs. BSC: 66 vs. 39 days, respectively (hazard ratio [HR] 0.31; 95%CI 0.11, 0.86; p = 0.009; FAS). Similar trends were observed for TTF and OS. CES2-positive patients had longer median PFS (158 vs. 56 days) and OS (228 vs. 82 days) vs. CES2-negative patients. Adverse events were predictable, dose-dependent and consistent with those previously observed with etoposide. These efficacy and safety findings in second-line BTC warrant further clinical investigation of CAP7.1.

Modelling the impact of detecting and treating ductal carcinoma in situ in a breast screening programme.

OBJECTIVES: Screening has substantially increased the detection of carcinoma in situ of the breast (CIS). Opinions vary as to whether this constitutes over-diagnosis or an opportunity to interrupt breast cancer's natural history. In England, incidence of invasive cancer and CIS increased in women of screening age (50-64 years), leading to a subsequent deficit in invasive incidence in women aged 65-69 years immediately beyond the invited age range. We aimed to model underlying incidence of invasive cancer and CIS expected in the absence of screening, and to quantify the likely relative contributions of their early detection to the observed reduction in invasive cancer in women of postscreening age. SETTING: UK NHS breast screening programme in England. METHODS: Poisson regression modelling was used to establish the underlying incidence of invasive and in situ cancers in the absence of screening. We then estimated age- and year-specific excess detection rates attributable to screening. Applying these to population figures we estimated conservatively the relative contributions of early diagnosis of CIS and invasive cancer at 50-64 years of age to the subsequent deficit in invasive cancer in women beyond invitation age (65-69 years), for screening early in the programme and at steady state. RESULTS: Our model estimated a 1.6% annual increase in incidence, giving an estimated deficit of 4.22 invasive cancers per 10,000 women aged 65-69 years in 1996. Carcinoma in situ contributed 13-17% to the deficit, assuming a mean six year lead time and 75-100% progression to invasive cancer. At steady state, with current screening performance and with lead times of 3-4 years (invasive cancer) and 6-9 years (CIS), invasive incidence might be reduced by 5-6 cancers per 10,000 women aged 65-69 years in 2010 (15-20% of underlying incidence), CIS contributing 20-40%. DISCUSSION: The longer lead time associated with CIS attenuates the impact its early detection has on subsequent invasive incidence. At steady state screening, its detection contributes significantly to the deficit in invasive incidence. Our results suggest that, cancer for cancer, there may be just as much benefit in detecting and treating a case of CIS as there is in treating a case of invasive cancer.

Cross-trial analysis of immunologic and clinical data resulting from phase I and II trials of MVA-5T4 (TroVax) in colorectal, renal, and prostate cancer patients.

The attenuated vaccinia virus MVA has been engineered to deliver the tumor antigen 5T4 (MVA-5T4; TroVax), a surface glycoprotein expressed by most solid tumors. MVA-5T4 has been tested in 2 phase I/II and 7 phase II clinical trials in colorectal (4 trials), renal (4 trials), and prostate (1 trial) advanced cancer patients. Data have been collated from all 9 studies and used to investigate the magnitude and kinetics of 5T4-specific antibody responses after vaccination and to identify potential associations between the immune response and patient survival. Antibody responses specific for the 5T4 tumor antigen and the MVA viral vector were quantified in plasma samples taken from cancer patients before and after the treatment with MVA-5T4. Immunologic and survival data were analyzed using proportional hazards regression adjusting for age and gender. Both survival and immunologic response data were available for 189 patients with colorectal (n=73), renal (n=89), and prostate (n=27) cancer. Before the treatment with MVA-5T4, 5T4-specific antibody levels were significantly elevated in cancer patients compared with healthy donors. After MVA-5T4 administration, 5T4-specific antibody responses increased significantly and peaked after 3 to 4 vaccinations. Exploratory analyses showed significant associations between 5T4 antibody responses and overall survival across all 9 trials and in patients with colorectal cancer. The 5T4-specific antibodies were present at higher levels in cancer patients compared with healthy donors and increased significantly after treatment with MVA-5T4. Although the studies were uncontrolled, there were encouraging signs of activity which is associated with the magnitude of 5T4-specific antibody responses.

The integration profile of EIAV-based vectors.

Lentiviral vectors based on equine infectious anemia virus (EIAV) stably integrate into dividing and nondividing cells such as neurons, conferring long-term expression of their transgene. The integration profile of an EIAV vector was analyzed in dividing HEK293T cells, alongside an HIV-1 vector as a control, and compared to a random dataset generated in silico. A multivariate regression model was generated and the influence of the following parameters on integration site selection determined: (a) within/not within a gene, (b) GC content within 20 kb, (c) within 10 kb of a CpG island, (d) gene density within a 2-Mb window, and (e) chromosome number. The majority of the EIAV integration sites (68%; n = 458) and HIV-1 integration sites (72%; n = 162) were within a gene, and both vectors favored AT-rich regions. Sites within genes were examined using a second model to determine the influence of the gene-specific parameters, gene region, and transcriptional activity. Both EIAV and HIV-1 vectors preferentially integrated within active genes. Unlike the gammaretrovirus MLV, EIAV and HIV-1 vectors do not integrate preferentially into the promoter region or the 5' end of the transcription unit.