Search

Background & aims Hepatic fibrosis secondary to HCV infection can lead to cirrhosis and hepatic decompensation. Sustained virologic response (SVR) is possible with direct-acting antiviral drug regimens; however, patients with advanced fibrosis have an increased risk for HCC. Heat shock protein 47 (HSP47), a key collagen chaperone, has been implicated in fibrosis development. Here, we evaluated the efficacy and safety of BMS-986263, a lipid nanoparticle delivering siRNA designed to degrade HSP47 mRNA, for the treatment of advanced fibrosis. Approach & results NCT03420768 was a Phase 2, randomized (1:1:2), placebo-controlled trial conducted at a hepatology clinic in the USA. Patients with HCV-SVR (for ≥1 year) and advanced fibrosis received once-weekly intravenous infusions of placebo or BMS-986263 (45 mg or 90 mg) for 12 weeks. The primary endpoint was ≥1 METAVIR stage improvement at Week 12; key secondary endpoints included Ishak score improvement, pharmacokinetics, fibrosis biomarkers, and safety. All 61 patients completed treatment and 2/15 (13%; placebo), 3/18 (17%; 45 mg), and 6/28 (21%; 90 mg) had METAVIR improvements of ≥1 stage at Week 12. Five patients in the 90 mg arm had Ishak improvements by ≥2 stages. BMS-986263 plasma concentrations increased in a generally dose-proportional fashion between BMS-986263 doses, with no notable accumulation with weekly dosing. All adverse events (AEs) were mild or moderate in intensity; most treatment-related AEs were infusion-related reactions in the BMS-986263 arms. At baseline, collagen levels were low, indicating low levels of fibrogenesis in these patients. Conclusions In patients with HCV-SVR, BMS-986263 administration was generally well tolerated through Week 36 and resulted in METAVIR and Ishak score improvements. Further evaluation of BMS-986263 in patients with active fibrogenesis is warranted.

Pre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes. The sera were tested for NAb in a Lec-2 cell line for AAV9 vector and in a COS-7 cell line for the other AAV serotypes. In the 60 Topigs-20 strain 2 to 4 years of age, 100% were positive for AAV2 NAb, 45% positive for AAV6 NAb, and ∼20% positive for each of AAV1, 5, 8, and 9 NAb. These data showed that ∼80% of Norsvin Topigs-20 pigs evaluated were seronegative for pre-existing NAb to the AAV1, 5, 8, and 9 serotypes, respectively. In 22 Gottingen minipigs at 5-6 months of age, serum AAV serotype-specific NAb coexisted with that of various other AAV serotypes at 32% to 46% between two serotypes. These results suggested that coexisting NAb resulted either from multiple AAV serotype coinfection or from one (or more) serotypes that can crossreact with other AAV serotypes in some minipigs. Among the 40 Yucatan minipigs, 20 of the minipigs were3 months old and were all negative for NAb against AAV5, 8, and 9, and only one of these 20 pigs was positive to AAV1 and 6. We further determined the titers in those positive pigs and found most Gottingen minipigs had low titer at 1:20, whereas some of Topigs-20 pigs had titers between 1:80 and 1:320, and some of Yucatan pigs had titers between 1:160 and 1:640. These results suggested that the majority of the pigs in the three strains would be amenable to gene therapy study using AAV1, AAV5, AAV8, and AAV9 and that prescreening on circulating AAV antibodies could be helpful before inclusion of pigs into studies.

Recombinant adeno associated viruses (rAAV) have become an important tool for the delivery of gene therapeutics due to long-standing safety and success in clinical trials. Since humans often become exposed to AAVs and develop anti-AAV antibodies (Abs), a potential impediment to the success of gene therapeutics is neutralization of the viral particle before it has had a chance to bind and enter target cells to release the transgene. Identification of subjects with preexisting Abs having neutralizing potential, and exclusion of such subjects from clinical studies is expected to enhance drug efficacy. In vitro cell-based reporter assays are most often employed to determine the level of neutralizing antibodies in a given population. Such assays measure the ability of the Abs to prevent viral binding and entry into cells by engaging epitopes on the viral capsid involved in host cell receptor binding. In general, cell-based assays are low throughput and labor intensive and may suffer from high variability and low sensitivity issues. In contrast, enzyme-linked immunosorbent assays (ELISAs) are simpler, less variable, and have higher throughput. Demonstrating a correlation between neutralizing Abs assessed by a cell-based assay and total binding Abs measured in an ELISA will enable the use and substitution of the latter for screening and exclusion of subjects. In this work, we describe the development of a highly sensitive, specific, robust, and reproducible chemiluminescent ELISA method for the detection of total anti-AAV9 Abs. Using this method, we analyzed the prevalence of preexisting anti-AAV9 Abs in 100 serum samples from heart disease patients. Analysis of neutralizing Abs in the same samples using an in vitro cell-based assay showed a strong correlation between total anti-AAV9 Abs and neutralizing Abs, indicating the feasibility of using the total Ab ELISA in the future for patient screening and exclusion.

The 2018 12th Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9–13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LC–MS, hybrid ligand binding assay (LBA)/LC–MS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC–MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC–MS for biotherapeutics and regulatory agencies’ inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.

Background: Orva-cel is an investigational B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell product genetically modified with a lentiviral vector to express a CAR construct with a unique fully human single-chain variable fragment, optimized spacer, and 4-1BB costimulatory and CD3ζ activation domains. Orva-cel is currently being evaluated for efficacy and safety in the ongoing phase 1/2 EVOLVE study (NCT03430011) in heavily pretreated patients with relapsed/refractory multiple myeloma. We characterized orva-cel drug products, manufactured using the process in place for the phase 2 portion of the study and intended for commercial manufacturing, for CAR+ T cell purity, phenotype, and function. Methods: Immunophenotyping was performed by flow cytometry of both surface and intracellular markers, including CD3, CD4, CD8, CD45, CCR7, CD45RA, CD28, CD27, and active caspase 3. Cytokine production after challenge with BCMA+ target cells was assessed by intracellular cytokine staining and Luminex multiplex assay of secreted cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrB). CAR-mediated in vitro proliferative capacity was measured after anti-idiotypic antibody stimulation using the IncuCyte Live-Cell Analysis System (Sartorius, Göttingen, Germany). In vivo CAR+ T cell proliferation and persistence were assessed by quantitative polymerase chain reaction (qPCR). Results: The orva-cel manufacturing process was designed to enable consistent production of highly pure CD3+ cell products (median frequency of CD3+ T cells, 99.96%; quartiles 1-3 interquartile range, 99.9%-100.0%; n = 81). Orva-cel drug products were characterized by high frequencies of less-differentiated CAR+ T cells, leading to a dominant central memory-like population (CCR7+CD45RA- CAR+ T cells) and substantial frequencies of naïve-like cells (CCR7+CD45RA+ CAR+ T cells) (Figure). When assayed for in vitro functional activity, orva-cel drug products showed robust antigen-specific cytokine and effector molecule production (IFN-γ, TNF-α, IL-2, and GrB) upon challenge with BCMA+ tumor cells, as well as vigorous proliferation in response to CAR stimulation. Preliminary correlative analysis suggested that the early memory phenotype may be linked to increased CAR+ T cell proliferative capacity, as determined by in vitro experiments and in vivo PK parameters (ie, maximum CAR+ T cell concentration observed in the blood [Cmax], time to Cmax, area under the curve from Day 0 to 28 [AUC0-28], and CAR+ T cell persistence at Month 3 and Month 6). Consistent with the early CAR T cell memory phenotype, qPCR analysis showed robust in vivo proliferation of CAR+ T cells after infusion, with a median Cmax of 1.54 × 105 transgene copies/µg DNA and median AUC0-28 of 1.61 × 106 transgene copies/µg DNA*day, as well as long-term in vivo persistence, with CAR+ T cells detected in 69% of patients at 6 months postinfusion. Conclusions: The orva-cel manufacturing process results in drug products characterized by highly pure T cells, with high frequencies of early memory and polyfunctional CAR+ T cells. Orva-cel drug products showed robust antigen-specific degranulation, production of multiple cytokines, sustained in vitro and in vivo proliferation, and in vivo persistence. Disclosures Colonna: Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Navarro:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Devries:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Beckett:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Amsberry:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Radhakrishnan:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Piasecki:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Heipel:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Li:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Kavita:Bristol Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Works:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Mujacic:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment.

ND-L02-s0201/BMS-986263 is a lipid nanoparticle (LNP) drug product containing a heat shock protein 47 (HSP47)–specific small interfering ribonucleic acid (siRNA) and being developed for the treatment of liver and idiopathic pulmonary fibrosis. To address immunogenicity-related issues, we developed a robust, fit-for-purpose (FFP) three-tier electrochemiluminescent (ECL) anti-drug antibody (ADA) assay for the detection of antibodies (Abs) generated to surface-exposed components of BMS-986263. The drug was coated directly on plates, and several Abs specific for polyethylene glycol (PEG) and other surface components were tested for use as positive quality controls (QCs). Following selection of a rabbit monoclonal anti-PEG Ab, the assay was optimized, and various method development challenges specific to the modality and pseudo surrogate rabbit control were addressed. Screening, confirmatory, and titer cut points were validated following a statistical evaluation of 41 individual K2EDTA human plasma samples at a minimum required dilution (MRD) of 100. Assay precision, sensitivity, selectivity, drug tolerance, and hook effect were determined for the rabbit Ab prepared in human K2EDTA plasma matrix. The assay was used to interrogate anti-drug Ab (ADA) responses in normal human subjects who were administered 90 mg of the drug intravenously (IV) once every week for 3 weeks in phase I clinical trials. All pre- and post-dose samples were found to be negative for ADA. Based on these results, we concluded that BMS-986263 is not immunogenic. To the best of our knowledge, this work represents the first ADA method developed and reported for an LNP-based drug product.

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.

The Gyrolab™ xP is a microfluidic platform for conducting ligand binding assays (LBAs) and is recognized for its utility in discovery bioanalysis. However, few reports have focused on the technology for regulated bioanalysis. This technology has the advantage of low reagent consumption, low sample volume, and automated ligand binding methods. To improve bioanalysis testing timelines and increase the speed at which biotherapeutics are delivered to patients, we evaluated the technology for its potential to deliver high-quality data at reduced testing timelines for regulated bioanalysis. Six LBA methods were validated to support bioanalysis for GLP toxicokinetic or clinical pharmacokinetic studies. Validation, sample analysis, and method transfer are described. In total, approximately 4000 samples have been tested for regulated bioanalysis to support 6 GLP toxicology studies and approximately 1000 samples to support 2 clinical studies. Gyrolab™ xP had high run pass rates (≥83%) and high incurred sample reanalysis (ISR) pass rates (>94%). The maximum total error observed across all QC levels for a given assay was

Background The ectodomain of matrix protein 2 (M2e) is a promising candidate for a broadly protective influenza A vaccine because it is highly conserved and antibodies to M2e are protective in animal models. STF2.4xM2e (VAX102) is a recombinant fusion protein that links four tandem copies of the M2e antigen to Salmonella typhimurium flagellin, a TLR5 ligand used as an adjuvant. The objectives of this first-in-human study were to assess the safety and immunogenicity of VAX102 given as a prime-boost regimen to healthy adults. Methods Sixty subjects 18–49 years old were enrolled in a multicenter, double-blind, randomized, placebo-controlled trial (Study 1). Based on pre-clincial data, initial design included doses starting at 10 μg, with an escalation plan. After reactogenicity was noted at the 10 μg dose, the trial was redesigned to evaluate 0.3, 1.0, and 3 μg doses. Following this study, 16 subjects were enrolled in Study 2, an open label, low dose study, to evaluate doses of 0.03 and 0.1 μg. In both trials, vaccine or placebo was given intramuscularly (i.m.) at 0 and 28 days. Clinical and laboratory safety assessments took place 1 and 7 days after immunization. Immune responses to M2e and flagellin were assessed by ELISA at 7, 14 and 28 days after each dose. Seroconversion was defined as a serum IgG anti-M2e antibody value ≥0.174 μg/ml and a fourfold rise in concentration. Results Doses of 0.03–1 μg were safe and well tolerated in all subjects. Doses of 0.03 and 0.1 μg produced limited immunogenicity (38% and 75% respectively), after the second dose of vaccine. Doses of 0.3 and 1.0 μg were immunogenic in 18 (75%) of 24 vaccinees after the first dose and 23 (96%) after the second dose. In the 1.0 μg group, the geometric mean M2e antibody concentration was 0.4 μg/ml after the first dose and 1.7 μg/ml after the booster dose. M2e antibody concentrations and seroconversion rates were not significantly different at higher doses ( p > 0.05). Immune response to flagellin was robust but did not appear to interfere with M2e antibody responses after the booster dose. Following the first injection of VAX102 at higher doses (3 and 10 μg), self-limited but severe symptoms were noted in some subjects and were associated with elevated levels of C-reactive protein. Although not directly measured, this reaction was believed to be mediated by cytokine release. Conclusions VAX102 was safe and induced high antibody levels to M2e at 0.3 and 1.0 μg doses. The TLR5 ligand, S. typhimurium flagellin, is a novel approach to adjuvant-like activity through activation of innate immunity, and when fused to multiple copies of the M2e protein, the vaccine was able to induce a fourfold rise in antibody in humans, to a previously non-immunogenic, highly-conserved portion of the influenza virus. Clinical correlates of protection that may be afforded by M2e antibody in humans are a future focus of investigation.

In the past few years, several studies have unravelled a novel pathway of antigen presentation to T cells of the mammalian immune system. The antigens are presented by CD1, which appears to have evolved to present glycolipid antigens to alphabeta T cells. CD1-restricted T cells are frequently autoreactive, and can promptly release key regulatory cytokines such as IL-4 and IFN-gamma. They have been implicated in a variety of autoimmune diseases including type I diabetes and lupus, in intracellular bacterial infections, and in tumor rejection. They are likely to be involved at the early, innate phase of these immune responses, providing a unique model to study the interface between the innate and adaptive immune systems.

In view of the observations that the calcium ionophores, A23187 and ionomycin, enhance the processing and secretion of interleukin-1 (IL-1 alpha) and IL-1 beta from macrophages, and IL-1 alpha processing is mediated by calpain, a calcium-dependent protease, we evaluated the possibility that calpain might also play a role in the processing of IL-1 beta. Whereas calpain-containing P388D1 macrophage lysates and purified calpain processed precursor IL-1 alpha to its mature 17-kDa form, precursor IL-1 beta was degraded by both sources of calpain. However, the activation of calpain in P388D1 cells that were transiently transfected with a cDNA expression vector encoding the precursor form of IL-1 beta did not result in the degradation of precursor IL-1 beta, but did result in the processing and secretion of IL-1 alpha, implying that precursor IL-1 beta is protected from calpain degradation in vivo. Furthermore, calpain did not enhance the processing of the IL-1 beta precursor by the IL-1 beta-converting enzyme. These results indicate that calpain is not involved in the processing of precursor IL-1 beta in vitro or in vivo. The IL-1 beta precursor may be protected from calpain degradation by a sequestering mechanism that involves a cytoplasmic factor(s) that reduces the sensitivity of IL-1 beta to attack by calpain or localizes IL-1 beta to a site that precludes any interaction with the protease. Although MDL 28,170, a calpain inhibitor, prevented the ionomycin-induced processing of precursor IL-1 alpha to the mature protein in P388D1 cells, it did not inhibit the ionomycin-induced secretion of the mature IL-1 alpha and -beta proteins expressed in these cells. These results indicate that a calcium-dependent factor other than calpain is involved in the secretion of the mature IL-1 proteins.

Background We evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand, flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin in VAX128B and was fused in both positions in VAX128C. Methods In a dose escalation trial, 112 healthy subjects 18–49 and 100 adults ≥65 years old were enrolled in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses ranging from 0.5 to 20 μg. VAX128C was selected for second study performed in 100 subjects 18–64 years old comparing 1.25 and 2.5 μg doses. All subjects were followed for safety and sera collected pre- and post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and cytokine levels were also measured. Conclusions In the first study high HAI titers and high seroconversion and seroprotection rates were observed at doses ≥2.5 μg in adults 18–49. In adults ≥65 years, the vaccines doses of ≥4 μg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8 μg, VAX128B to 16 μg and VAX128C to 20 μg. Dose escalation for VAX128A was stopped at 8 μg because one subject had temperature 101.6 °F associated with a high CRP response, VAX128B was stopped at 16 μg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20 μg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5 μg. The peak GMT was 385 (95%CI 272–546), 79% (71–87%) seroconversion and 92% (84–96%) seroprotection. Discussion Flagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology.

Influenza vaccines are less effective in older people than younger people. This impaired ability to protect older people from influenza viral lung infection has important implications as older people suffer a higher morbidity and mortality from influenza viral lung infection than younger people. Therefore, the development of novel effective vaccines that induce protection from influenza viral infections in older people are urgently needed. We had previously shown that direct linking the TLR5 activator, flagellin, to viral peptides induces effective immunity to viral antigens in young mice and people, respectively. In this study, we tested the efficacy of this vaccine platform with the hemagglutinin peptide of the influenza A strain virus (vaccine denoted as STF2.HA1-2) in protecting aged mice from subsequent influenza viral lung infection. We found that a 3.0 μg dose of the vaccine was effective in reducing mortality and increasing clinical well-being during influenza viral lung infection in aged mice. However, this effect was inferior to the response induced in young mice. Defects in the adaptive immune system but not the innate immune system were associated with this reduced effectiveness of the vaccine with aging. Our results indicate that the STF2.HA1-2 vaccine is effective in protecting aged hosts from influenza lung infection, although defects in the adaptive immune system with aging may limit the effectiveness of this vaccine in older people.

Background Influenza vaccines perform poorly in the elderly with reduced serological response and vaccine efficacy. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in Escherichia coli. Methods 120 subjects ≥65 years old were enrolled at three clinical centers. VAX125 vaccine was administered at doses of 0.5, 1, 2, 3, 5 or 8 μg delivered i.m. as a single dose vaccination on Day 0 using a dose-escalation with 20 subjects in each dose level. Subjects were followed for adverse events and sera were tested by hemagglutination-inhibition (HAI) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP) and anti-flagellin antibody were also assessed. Results The mean age was 71 years. The vaccine was well tolerated at all dose levels, with no more than mild to moderate local or systemic symptoms. The geometric mean titers (GMT) increased in all dose groups. In the 5 μg group the day 14 post-vaccination HAI titer was 1:226 showing a 12-fold increase over baseline. The 8 μg group showed a similar post-vaccination GMT increase (∼8-fold). In the combined 5 and 8 μg groups, the seroconversion rate was 75% and the seroprotection rate was 98%. Conclusions A 5 μg dose of VAX125 was safe and able to induce a greater than 10-fold increase HAI antibody levels and nearly complete seroprotection in subjects over 65 years old. The use of flagellin to adjuvant influenza vaccines via the TLR5 innate immune pathway appears to be a useful approach to overcome poor immune responses in the elderly. VAX125 is a promising new candidate for prevention of influenza A disease in both young adults and the elderly.

Background Currently controversy exists about the immunogenicity of seasonal trivalent influenza vaccine in certain populations, especially the elderly. STF2.4×M2e (VAX102) is a recombinant fusion protein that links four copies of the ectodomain of influenza virus matrix protein 2 (M2e) antigen to Salmonella typhimurium flagellin, a TLR5 ligand. The objectives of this study were to assess the feasibility of giving VAX102 and TIV in combination in an effort to achieve greater immunogenicity and to provide cross-protection. Methodology/Principal Findings Eighty healthy subjects, 18-49 years old, were enrolled in May and June 2009 in a double-blind, randomized, controlled trial at two clinical sites. Subjects were randomized to receive either TIV + VAX102 or TIV + placebo. Both arms tolerated the vaccines. Pain at the injection site was more severe with TIV + VAX102. Two weeks after immunization the HAI responses to the H1 and H3 antigens of TIV were higher in those that received TIV + VAX102 than in TIV + placebo (309 vs 200 and 269 vs 185, respectively), although statistically non-significant. There was no difference in the HAI of the B antigen. In the TIV + VAX102 arm, the geometric mean M2e antibody concentration was 0.5 µg/ml and 73% seroconverted. Conclusions/Significance The combination of TIV + VAX102 has the potential to increase the immune response to the influenza A components of TIV and to provide M2e immunity which may protect against influenza A strains not contained in seasonal TIV. Trial Registration ClinicalTrials.gov NCT00921973

Background The need for worldwide seasonal and pandemic vaccine production has increased interest in the development of innovative technologies for influenza vaccine production. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in E. coli. Methods 128 healthy adult subjects 18–49 years old were enrolled in a clinical trial conducted in three stages at a single center. Stage 1 was an open-label, dose escalation study in which the VAX125 vaccine was administered intramuscularly (im) at doses of 0.1 μg, 0.3 μg, 1 μg, 2 μg, 3 μg, 5 μg and 8 μg to groups of 8 subjects each. Stage 2 was a double-blind, placebo-controlled study in which subjects were randomized to receive 1.0 μg and 2.0 μg VAX125 vaccine doses or placebo, with 16 subjects per group. Finally, an additional 24 subjects received a 0.5 μg dose of VAX125 in stage 3, which was a non-randomized, open label study. In all parts subjects were followed for adverse events and sera was tested by hemagglutination-inhibition (HAI) and microneutralization (MN) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP), cytokine levels, and anti-flagellin antibody were also assessed. Results Vaccine was generally well tolerated and there were no serious adverse events. Pain at the injection site was the most common local adverse event, and was mild or moderate in intensity. Systemic symptoms after vaccination include fatigue and headache, and two subjects, who received either 3 or 8 μg, had moderately severe systemic symptoms accompanied by substantial increases in serum CRP. Serum antibody responses against SI were seen by HAI and MN in most study subjects, with the geometric mean titer of post vaccination antibody increasing in a dose-dependent fashion. Overall, four-fold or greater serum HAI responses were seen in 61 of 96 (64%) subjects who received doses of 0.5 μg or greater, including in 46 of 72 subjects who received doses from 0.5 μg to 2 μg. Conclusions The globular head of the influenza HA expressed in a prokaryotic system was able to induce a functional antibody response against native virions. Vigorous responses were seen at relatively low doses of HA antigen suggesting that the addition of flagellin provided a substantial adjuvanting effect. The high levels of immune response at low doses of antigen and the relative ease of production associated with E. coli expression suggests that this approach may represent an effective strategy for enhancing the global influenza vaccine supply.

Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model.

Mouse CD1.1 is an MHC class I-like, non-MHC-encoded, surface glycoprotein that can be recognized by T cells, in particular NK1.1+ T cells, a subset of αβ T cells with semiinvariant TCRs that promptly releases potent cytokines such as IL-4 and IFN-γ upon stimulation. To gain insight into the function of CD1.1, a panel of nine mAbs was generated and used to biochemically characterize and monitor the surface expression of CD1.1 on different cell types. CD1.1 is a heavily glycosylated, β2-microglobulin-associated surface protein. Its recognition by a panel of 12 Vα14-positive and -negative CD1-specific αβ T cell hybridomas was blocked by two groups of mAbs that bound to adjacent clusters of epitopes, indicating that different αβ TCRs bind to the same region of CD1.1, presumably above the groove. Remarkably, CD1.1 was mainly expressed by dendritic cells, B cells, and macrophages, suggesting a function in Ag presentation to Th cells. Furthermore, the cell type that expressed the highest levels of CD1.1 was the splenic marginal zone B cell, a distinct subset of B cells that also expresses CD21 (the C3d receptor) and may be involved in natural responses to bacterial Ags. Altogether, the results support the idea that CD1.1 may function in recruiting a form of innate help from specialized cytokine producer αβ T cells to APCs, a role that might be important at the preadaptive phase of immune responses to some microbial pathogens.