Diagnostic journey for individuals with fibrous dysplasia / McCune albright syndrome (FD/MAS).

BACKGROUND: Reducing delayed diagnosis is a significant healthcare priority for individuals with rare diseases. Fibrous Dysplasia/ McCune Albright Syndrome (FD/MAS) is a rare bone disease caused by somatic activation mutations of NASA. FD/MAS has a broad clinical phenotype reflecting variable involvement of bone, endocrine and other tissues, distribution and severity. The variable phenotype is likely to prolong the diagnostic journey for patients further. AIM: To describe the time from symptom onset to final diagnosis in individuals living with FDMAS. METHODS: We used the UK-based RUDY research database ( www.rudystudy.org ), where patients self-report their diagnosis of FD/MAS. Participants are invited to complete the diagnostic journey based on the EPIRARE criteria. RESULTS: 51 individuals diagnosed with FD/MAS were included in this analysis. Among them, 70% were female, and the median age was 51.0 years (IQR 34.5-57.5]. 12 (35%) individuals reported McCune Albright Syndrome, 11 (21.6%) craniofacial and 11(21.6%) for each of poly- and mono-ostotic FD and 6 (11.8%) did not know their type of FD/MAS. Pain was the commonest first symptom (58.8%), and 47.1% received another diagnosis before the diagnosis of FD/MAS. The median time to final diagnosis from the first symptom was two years with a wide IQR (1,18) and range (0-59 years). Only 12 (23.5%) of individuals were diagnosed within 12 months of their first symptoms. The type of FD/MAS was not associated with the reported time to diagnosis. Significant independent predictors of longer time to final diagnosis included older current age, younger age at first symptom and diagnosis after 2010. CONCLUSION: Individuals with FDMAS have a variable time to diagnosis that can span decades. This study highlights the need for further research on how to improve diagnostic pathways within Orthopaedic and Ear, Nose and Throat (ENT)/Maxillofacial services. Our data provides a baseline to assess the impact of novel NHS diagnostic networks on reducing the diagnostic odyssey.

Longitudinal dynamics and clinically available predictors of poor response to COVID-19 vaccination in multiple myeloma.

Not available.

Determinants of durable humoral and T cell immunity in myeloma patients following COVID-19 vaccination.

OBJECTIVE: To describe determinants of persisting humoral and cellular immune response to the second COVID-19 vaccination among patients with myeloma. METHODS: This is a prospective, observational study utilising the RUDYstudy.org platform. Participants reported their second and third COVID-19 vaccination dates. Myeloma patients had an Anti-S antibody level sample taken at least 21 days after their second vaccination and a repeat sample before their third vaccination. RESULTS: 60 patients provided samples at least 3 weeks (median 57.5 days) after their second vaccination and before their third vaccination (median 176.0 days after second vaccine dose). Low Anti-S antibody levels (<50 IU/mL) doubled during this interval (p = .023) and, in the 47 participants with T-spot data, there was a 25% increase negative T-spot tests (p = .008). Low anti-S antibody levels prior to the third vaccination were predicted by lower Anti-S antibody level and negative T-spot status after the second vaccine. Independent determinants of a negative T-spot included increasing age, previous COVID infection, high CD4 count and lower percentage change in Anti-S antibody levels. CONCLUSIONS: Negative T-spot results predict low Anti-S antibody levels (<50 IU/mL) following a second COVID-19 vaccination and a number of biomarkers predict T cell responses in myeloma patients.

Targeting myeloid chemotaxis to reverse prostate cancer therapy resistance.

Inflammation is a hallmark of cancer1. In patients with cancer, peripheral blood myeloid expansion, indicated by a high neutrophil-to-lymphocyte ratio, associates with shorter survival and treatment resistance across malignancies and therapeutic modalities2-5. Whether myeloid inflammation drives progression of prostate cancer in humans remain unclear. Here we show that inhibition of myeloid chemotaxis can reduce tumour-elicited myeloid inflammation and reverse therapy resistance in a subset of patients with metastatic castration-resistant prostate cancer (CRPC). We show that a higher blood neutrophil-to-lymphocyte ratio reflects tumour myeloid infiltration and tumour expression of senescence-associated mRNA species, including those that encode myeloid-chemoattracting CXCR2 ligands. To determine whether myeloid cells fuel resistance to androgen receptor signalling inhibitors, and whether inhibiting CXCR2 to block myeloid chemotaxis reverses this, we conducted an investigator-initiated, proof-of-concept clinical trial of a CXCR2 inhibitor (AZD5069) plus enzalutamide in patients with metastatic CRPC that is resistant to androgen receptor signalling inhibitors. This combination was well tolerated without dose-limiting toxicity and it decreased circulating neutrophil levels, reduced intratumour CD11b+HLA-DRloCD15+CD14- myeloid cell infiltration and imparted durable clinical benefit with biochemical and radiological responses in a subset of patients with metastatic CRPC. This study provides clinical evidence that senescence-associated myeloid inflammation can fuel metastatic CRPC progression and resistance to androgen receptor blockade. Targeting myeloid chemotaxis merits broader evaluation in other cancers.

A Phase I Trial of CT900, a Novel α-Folate Receptor-Mediated Thymidylate Synthase Inhibitor, in Patients with Solid Tumors with Expansion Cohorts in Patients with High-Grade Serous Ovarian Cancer.

PURPOSE: CT900 is a novel small molecule thymidylate synthase inhibitor that binds to α-folate receptor (α-FR) and thus is selectively taken up by α-FR-overexpressing tumors. PATIENTS AND METHODS: A 3+3 dose escalation design was used. During dose escalation, CT900 doses of 1-6 mg/m2 weekly and 2-12 mg/m2 every 2 weeks (q2Wk) intravenously were evaluated. Patients with high-grade serous ovarian cancer were enrolled in the expansion cohorts. RESULTS: 109 patients were enrolled: 42 patients in the dose escalation and 67 patients in the expansion cohorts. At the dose/schedule of 12 mg/m2/q2Wk (with and without dexamethasone, n = 40), the most common treatment-related adverse events were fatigue, nausea, diarrhea, cough, anemia, and pneumonitis, which were predominantly grade 1 and grade 2. Levels of CT900 more than 600 nmol/L needed for growth inhibition in preclinical models were achieved for >65 hours at a dose of 12 mg/m2. In the expansion cohorts, the overall response rate (ORR), was 14/64 (21.9%). Thirty-eight response-evaluable patients in the expansion cohorts receiving 12 mg/m2/q2Wk had tumor evaluable for quantification of α-FR. Patients with high or medium expression had an objective response rate of 9/25 (36%) compared with 1/13 (7.7%) in patients with negative/very low or low expression of α-FR. CONCLUSIONS: The dose of 12 mg/m2/q2Wk was declared the recommended phase II dose/schedule. At this dose/schedule, CT900 exhibited an acceptable side effect profile with clinical benefit in patients with high/medium α-FR expression and warrants further investigation.

Phase 1, dose-escalation study of guadecitabine (SGI-110) in combination with pembrolizumab in patients with solid tumors.

BACKGROUND: Data suggest that immunomodulation induced by DNA hypomethylating agents can sensitize tumors to immune checkpoint inhibitors. We conducted a phase 1 dose-escalation trial (NCT02998567) of guadecitabine and pembrolizumab in patients with advanced solid tumors. We hypothesized that guadecitabine will overcome pembrolizumab resistance. METHODS: Patients received guadecitabine (45 mg/m2 or 30 mg/m2, administered subcutaneously on days 1-4), with pembrolizumab (200 mg administered intravenously starting from cycle 2 onwards) every 3 weeks. Primary endpoints were safety, tolerability and maximum tolerated dose; secondary and exploratory endpoints included objective response rate (ORR), changes in methylome, transcriptome, immune contextures in pre-treatment and on-treatment tumor biopsies. RESULTS: Between January 2017 and January 2020, 34 patients were enrolled. The recommended phase II dose was guadecitabine 30 mg/m2, days 1-4, and pembrolizumab 200 mg on day 1 every 3 weeks. Two dose-limiting toxicities (neutropenia, febrile neutropenia) were reported at guadecitabine 45 mg/m2 with none reported at guadecitabine 30 mg/m2. The most common treatment-related adverse events (TRAEs) were neutropenia (58.8%), fatigue (17.6%), febrile neutropenia (11.8%) and nausea (11.8%). Common, grade 3+ TRAEs were neutropaenia (38.2%) and febrile neutropaenia (11.8%). There were no treatment-related deaths. Overall, 30 patients were evaluable for antitumor activity; ORR was 7% with 37% achieving disease control (progression-free survival) for ≥24 weeks. Of 12 evaluable patients with non-small cell lung cancer, 10 had been previously treated with immune checkpoint inhibitors with 5 (42%) having disease control ≥24 weeks (clinical benefit). Reduction in LINE-1 DNA methylation following treatment in blood (peripheral blood mononuclear cells) and tissue samples was demonstrated and methylation at transcriptional start site and 5' untranslated region gene regions showed enriched negative correlation with gene expression. Increases in intra-tumoural effector T-cells were seen in some responding patients. Patients having clinical benefit had high baseline inflammatory signature on RNAseq analyses. CONCLUSIONS: Guadecitabine in combination with pembrolizumab is tolerable with biological and anticancer activity. Reversal of previous resistance to immune checkpoint inhibitors is demonstrated.

Immune response to COVID-19 vaccination is attenuated by poor disease control and antimyeloma therapy with vaccine driven divergent T-cell response.

Myeloma patients frequently respond poorly to bacterial and viral vaccination. A few studies have reported poor humoral immune responses in myeloma patients to COVID-19 vaccination. Using a prospective study of myeloma patients in the UK Rudy study cohort, we assessed humoral and interferon gamma release assay (IGRA) cellular immune responses to COVID-19 vaccination post second COVID-19 vaccine administration. We report data from 214 adults with myeloma (n = 204) or smouldering myeloma (n = 10) who provided blood samples at least three weeks after second vaccine dose. Positive Anti-spike antibody levels (> 50 iu/ml) were detected in 189/203 (92.7%), positive IGRA responses were seen in 97/158 (61.4%) myeloma patients. Only 10/158 (6.3%) patients were identified to have both a negative IGRA and negative anti-spike protein antibody response. In all, 95/158 (60.1%) patients produced positive results for both anti-spike protein serology and IGRA. After adjusting for disease severity and myeloma therapy, poor humoral immune response was predicted by male gender. Predictors of poor IGRA included anti-CD38/anti-BCMA (B-cell maturation antigen) therapy and Pfizer-BioNTech vaccination. Further work is required to understand the clinical significance of divergent cellular response to vaccination.

On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation.

The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of 'on-target hexamerisation', whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF.

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.

Triplet Therapy with Palbociclib, Taselisib, and Fulvestrant in PIK3CA-Mutant Breast Cancer and Doublet Palbociclib and Taselisib in Pathway-Mutant Solid Cancers.

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.

Intermittent schedules of the oral RAF-MEK inhibitor CH5126766/VS-6766 in patients with RAS/RAF-mutant solid tumours and multiple myeloma: a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study.

BACKGROUND: CH5126766 (also known as VS-6766, and previously named RO5126766), a novel MEK-pan-RAF inhibitor, has shown antitumour activity across various solid tumours; however, its initial development was limited by toxicity. We aimed to investigate the safety and toxicity profile of intermittent dosing schedules of CH5126766, and the antitumour activity of this drug in patients with solid tumours and multiple myeloma harbouring RAS-RAF-MEK pathway mutations. METHODS: We did a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study at the Royal Marsden National Health Service Foundation Trust (London, UK). Patients were eligible for the study if they were aged 18 years or older, had cancers that were refractory to conventional treatment or for which no conventional therapy existed, and if they had a WHO performance status score of 0 or 1. For the dose-escalation phase, eligible patients had histologically or cytologically confirmed advanced or metastatic solid tumours. For the basket dose-expansion phase, eligible patients had advanced or metastatic solid tumours or multiple myeloma harbouring RAS-RAF-MEK pathway mutations. During the dose-escalation phase, we evaluated three intermittent oral schedules (28-day cycles) in patients with solid tumours: (1) 4·0 mg or 3·2 mg CH5126766 three times per week; (2) 4·0 mg CH5126766 twice per week; and (3) toxicity-guided dose interruption schedule, in which treatment at the recommended phase 2 dose (4·0 mg CH5126766 twice per week) was de-escalated to 3 weeks on followed by 1 week off if patients had prespecified toxic effects (grade 2 or worse diarrhoea, rash, or creatinine phosphokinase elevation). In the basket dose-expansion phase, we evaluated antitumour activity at the recommended phase 2 dose, determined from the dose-escalation phase, in biomarker-selected patients. The primary endpoints were the recommended phase 2 dose at which no more than one out of six patients had a treatment-related dose-limiting toxicity, and the safety and toxicity profile of each dosing schedule. The key secondary endpoint was investigator-assessed response rate in the dose-expansion phase. Patients who received at least one dose of the study drug were evaluable for safety and patients who received one cycle of the study drug and underwent baseline disease assessment were evaluable for response. This trial is registered with ClinicalTrials.gov, NCT02407509. FINDINGS: Between June 5, 2013, and Jan 10, 2019, 58 eligible patients were enrolled to the study: 29 patients with solid tumours were included in the dose-escalation cohort and 29 patients with solid tumours or multiple myeloma were included in the basket dose-expansion cohort (12 non-small-cell lung cancer, five gynaecological malignancy, four colorectal cancer, one melanoma, and seven multiple myeloma). Median follow-up at the time of data cutoff was 2·3 months (IQR 1·6-3·5). Dose-limiting toxicities included grade 3 bilateral retinal pigment epithelial detachment in one patient who received 4·0 mg CH5126766 three times per week, and grade 3 rash (in two patients) and grade 3 creatinine phosphokinase elevation (in one patient) in those who received 3·2 mg CH5126766 three times per week. 4·0 mg CH5126766 twice per week (on Monday and Thursday or Tuesday and Friday) was established as the recommended phase 2 dose. The most common grade 3-4 treatment-related adverse events were rash (11 [19%] patients), creatinine phosphokinase elevation (six [11%]), hypoalbuminaemia (six [11%]), and fatigue (four [7%]). Five (9%) patients had serious treatment-related adverse events. There were no treatment-related deaths. Eight (14%) of 57 patients died during the trial due to disease progression. Seven (27% [95% CI 11·6-47·8]) of 26 response-evaluable patients in the basket expansion achieved objective responses. INTERPRETATION: To our knowledge, this is the first study to show that highly intermittent schedules of a RAF-MEK inhibitor has antitumour activity across various cancers with RAF-RAS-MEK pathway mutations, and that this inhibitor is tolerable. CH5126766 used as a monotherapy and in combination regimens warrants further evaluation. FUNDING: Chugai Pharmaceutical.

Phase I Trial of the PARP Inhibitor Olaparib and AKT Inhibitor Capivasertib in Patients with BRCA1/2- and Non-BRCA1/2-Mutant Cancers.

Preclinical studies have demonstrated synergy between PARP and PI3K/AKT pathway inhibitors in BRCA1 and BRCA2 (BRCA1/2)-deficient and BRCA1/2-proficient tumors. We conducted an investigator-initiated phase I trial utilizing a prospective intrapatient dose- escalation design to assess two schedules of capivasertib (AKT inhibitor) with olaparib (PARP inhibitor) in 64 patients with advanced solid tumors. Dose expansions enrolled germline BRCA1/2-mutant tumors, or BRCA1/2 wild-type cancers harboring somatic DNA damage response (DDR) or PI3K-AKT pathway alterations. The combination was well tolerated. Recommended phase II doses for the two schedules were: olaparib 300 mg twice a day with either capivasertib 400 mg twice a day 4 days on, 3 days off, or capivasertib 640 mg twice a day 2 days on, 5 days off. Pharmacokinetics were dose proportional. Pharmacodynamic studies confirmed phosphorylated (p) GSK3ß suppression, increased pERK, and decreased BRCA1 expression. Twenty-five (44.6%) of 56 evaluable patients achieved clinical benefit (RECIST complete response/partial response or stable disease ≥ 4 months), including patients with tumors harboring germline BRCA1/2 mutations and BRCA1/2 wild-type cancers with or without DDR and PI3K-AKT pathway alterations. SIGNIFICANCE: In the first trial to combine PARP and AKT inhibitors, a prospective intrapatient dose- escalation design demonstrated safety, tolerability, and pharmacokinetic-pharmacodynamic activity and assessed predictive biomarkers of response/resistance. Antitumor activity was observed in patients harboring tumors with germline BRCA1/2 mutations and BRCA1/2 wild-type cancers with or without somatic DDR and/or PI3K-AKT pathway alterations.This article is highlighted in the In This Issue feature, p. 1426.

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration.

Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).

Generation and Production of Modified Vaccinia Virus Ankara (MVA) as a Vaccine Vector.

The smallpox vaccine based on the vaccinia virus was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one to two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is an attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. MVA can encode one or more foreign antigens and thus can function as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant properties, and induces humoral and cellular immune responses. Many clinical trials of these new vaccines have been conducted, and the safety of MVA is now well documented. Immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate. In this chapter, we provide protocols for generation, isolation, amplification, and purification of recombinant MVA for preclinical and clinical evaluation.

Health-related quality of life and a cost-utility simulation of adults in the UK with osteogenesis imperfecta, X-linked hypophosphatemia and fibrous dysplasia.

BACKGROUND: Health-related quality of life of adults with osteogenesis imperfecta (OI), fibrous dysplasia (FD) and X-linked hypophosphatemia (XLH) remains poorly described. The aim of this study was to describe the HRQoL of adults with osteogenesis imperfecta, fibrous dysplasia and X-linked hypophophataemia and perform a cost-utility simulation to calculate the maximum cost that a health care system would be willing to pay for a hypothetical treatment of a rare bone disease. RESULTS: Participants completed the EQ-5D-5 L questionnaire between September 2014 and March 2016. For the economic simulation, we considered a hypothetical treatment that would be applied to OI participants in the lower tertile of the health utility score. A total of 109 study participants fully completed the EQ-5D-5 L questionnaire (response rate 63%). Pain/discomfort was the most problematic domain for participants with all three diseases (FD 31%, XLH 25%, OI 16%). The economic simulation identified an expected treatment impact of +2.5 QALYs gained per person during the 10-year period, which led to a willing to pay of £14,355 annually for a health care system willing to pay up to £50,000 for each additional QALY gained by an intervention. CONCLUSIONS: This is the first study to quantitatively measure and compare the HRQoL of adults with OI, FD and XLH and the first to use such data to conduct an economic simulation leading to healthcare system willingness-to-pay estimates for treatment of musculoskeletal rare diseases at various cost-effectiveness thresholds.

Laboratory-Scale Production of Replication-Deficient Adenovirus Vectored Vaccines.

Replication-deficient adenoviruses are potent vaccine development platforms used extensively for human and animal candidate vaccines, largely due to their very good safety and immunogenicity profile. In this chapter we describe a method that can be used in any laboratory for the scalable production of replication-deficient adenovirus vector vaccines to GLP for preclinical studies in animal models, including definitive experimental studies in large target animal species for veterinary applications. We use human adenovirus serotype 5 (HAdV5) as an example, but the method can be easily adapted for use with other adenovirus serotypes from different species of origin.

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice.

BACKGROUND: Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. METHODS: Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. RESULTS: A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. CONCLUSIONS: Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

Towards a universal vaccine for avian influenza: protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus.

Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP+M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP+M1 and a secondary vaccination with MVA-NP+M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry.

Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors.

First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture.