Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata).

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Overcoming the challenges in diagnosis of AQP4-IgG positive neuromyelitis optica spectrum disorders in resource poor settings using an indigenized and cost effective cell based assay.

BACKGROUND: Diagnosis of neuromyelitis optica spectrum disorders (NMOSD) in India is hindered by limited access to cost effective and sensitive assays for detection of aquaporin-4 antibody (AQP4-IgG) in India. OBJECTIVE: To develop a cost effective, sensitive, cell based assay (CBA) for detection of AQP4-IgG and to evaluate the serological status in patients with NMOSD diagnosed by 2015 diagnostic criteria. METHOD: Stably transfected Chinese hamster ovary (CHO) cell line expressing aquaporin M23 isomer was established. A fixed CBA was developed and validated in 381 samples including clinically definite NMOSD (n = 87), high risk NMOSD (n = 51), other demyelinating disorders (n = 92), other neurological disorders (n = 51) and healthy volunteers (n = 100). We tested the same samples again using a commercially available CBA and compared the results. All assays were performed by 2 independent investigators blinded to clinical and serological status. RESULTS: Our "in house"(Mangalore) assay showed sensitivity of 81.6% (95% CI 71.86-89.11%) for clinically definite NMOSD and 29.41% (95% CI 17.50-43.8%) for high risk NMOSD. Specificity was 100% for both groups. Both assays showed similar results for 67/ 87 (77.01%) patients with definite NMOSD while 4 samples tested positive by our assay alone (Cohen's kappa coefficient [K] - 0.86). Among the high risk group 14/51 (27.5%) samples showed similar results, one patient additionally was positive by the Mangalore assay (K - 0.95).

A Low-Cost SARS-CoV-2 rRBD ELISA to Detect Serostatus in Ecuadorian Population with COVID-19.

Laboratory diagnosis of the COVID-19 relies on RT-PCR to amplify specific fragments of SARS-CoV-2 genome. However, serological tests are required to determine the immune response elicited after infection. Here, we analyzed convalescent sera collected from positive individuals by RT-PCR to SARS-CoV-2 (n = 78), Zika (n = 20), dengue (n = 20), chikungunya (n = 54), intestinal parasites (n = 11), and HIV (n = 1), from different areas of Ecuador, with an in-house ELISA using a SARS-CoV-2 receptor binding domain recombinant (rRBD) antigen to detect IgG antibodies elicited by SARS-CoV-2 infection. Of the 78 samples positive for SARS-CoV-2 by RT-PCR, 73 showed high absorbance value compared with the cutoff and five were negative. All tested sera from other infections showed no reactivity. Sensitivity, specificity, positive predictive value, and negative predictive value were 93.6%, 100%, 100%, and 95.4%, respectively. This in-house anti-IgG rRBD ELISA offers an economic and simple alternative to determine IgG immune responses after SARS-CoV-2 infection.

E. coli expression and immunological assessment of expressed recombinant Newcastle disease virus hemagglutinin-neuraminidase protein in chickens.

Every year, the poultry industry experiences significant economic losses due to epidemics of Newcastle disease virus (NDV). Developing new vaccines by identifying and using the immunogenic hemagglutinin-neuraminidase (HN) protein can protect the poultry industry. In the present study, the full-length HN protein was expressed in Escherichia coli (E. coli) BL21 (DE3) cells, purified via affinity chromatography and detected via western blot analysis using His-specific antibodies. The purified HN protein was further evaluated in chickens to study the immune response against NDV. The successful production of HN-specific IgY proved the activity of the purified HN protein. IgY was present in the serum of immunized chickens. However, the immune response was higher in chickens immunized with purified HN protein along with complete and incomplete adjuvants than in chickens immunized with only the HN protein. Keywords: protein; Newcastle disease virus; poultry; infectious diseases; vaccines.

A new recombinant F1 antigen as a cost and time-effective tool for plague diagnosis.

The Yersinia pestis capsular antigen F1 is widely used in plague laboratory diagnosis. Here, we describe the production of an F1 recombinant protein within reduced time and biosafety requirements. Its evaluation in hemagglutination tests indicated that the recombinant F1 can replace the conventional F1 protein for plague diagnosis.

Immunogenicity Risk Assessment for an Engineered Human Cytokine Analogue Expressed in Different Cell Substrates.

The purpose of this article is to illustrate how performance of an immunogenicity risk assessment at the earliest stage of product development can be instructive for critical early decision-making such as choice of host system for expression of a recombinant therapeutic protein and determining the extent of analytical characterization and control of heterogeneity in co- and post-translational modifications. Application of a risk-based approach for a hypothetical recombinant DNA analogue of a human endogenous cytokine with immunomodulatory functions is described. The manner in which both intrinsic and extrinsic factors could interact to influence the relative scale of risk associated with expression in alternative hosts, namely Chinese hamster ovary (CHO) cells, Pichia pastoris, Escherichia coli, or Nicotinia tabacum is considered in relation to the development of the investigational product to treat an autoimmune condition. The article discusses how particular product-related variants (primary amino acid sequence modifications and post-translational glycosylation or other modifications) and process-derived impurities (host cell proteins, endotoxins, beta-glucans) associated with the different expression systems might influence the impact of immunogenicity on overall clinical benefit versus risk for a therapeutic protein candidate that has intrinsic MHC Class II binding potential. The implications of the choice of expression system for relative risk are discussed in relation to specific actions for evaluation and measures for risk mitigation, including use of in silico and in vitro methods to understand intrinsic immunogenic potential relative to incremental risk associated with non-human glycan and protein impurities. Finally, practical guidance on presentation of this information in regulatory submissions to support clinical development is provided.

Prophylaxis therapy with bypassing agents in patients with haemophilia A and inhibitors undergoing surgery: A cost analysis in Spain.

OBJECTIVES: This study estimated the cost of prophylaxis with activated prothrombin complex concentrate (aPCC) and recombinant activated factor VIIa (rFVIIa) in surgical patients with haemophilia A and inhibitors in Spain. METHODS: A decision-analytic model was developed to estimate the cost to the Spanish National Health System of providing haemostatic coverage in this haemophilia population, with age distribution and average weight derived from the literature, and the annual number of surgeries (0.33 per patient) from local data. Drug costs were calculated from official ex-factory prices with a 7.5% mandatory deduction and recommended dosing regimens. RESULTS: The estimated average costs per patient were €10 100.73 (aPCC) and €14 265.89 (rFVIIa) for dental extraction, €24 043.88 (aPCC) and €62 301.08 (rFVIIa) for minor surgery and €126 595.81 (aPCC) and €347 731.09 (rFVIIa) for major surgery. Assuming an estimated 23 annual surgeries in this population (N = 69), distributed as 19% dental extraction, 50% minor surgery and 31% major surgery, the total annual cost of prophylaxis was €1 209 682.35 with aPCC and €3 221 929.28 with rFVIIa. CONCLUSIONS: aPCC costs were 62.5% lower than rFVIIa. Assuming potential clinical equivalence, aPCC is a potentially cost-saving option for surgical patients with haemophilia A and inhibitors.

Development of a biochemical and biophysical suite for integral membrane protein targets: A review.

The generation of integral membrane proteins (IMPs) in heterologous systems and their characterization remains a major challenge in biomedical research. Significant efforts have been invested both in academia and in the pharmaceutical industry to establish technologies for the expression, isolation and characterization of IMPs. Here we summarize some of the key aspects, which are important to support structure-based drug design (SBDD) in drug discovery projects. We furthermore include timeline estimates and an overview of the target selection and biophysical screening approaches.

In silico selection of functionally important proteins from the mialome of Ornithodoros erraticus ticks and assessment of their protective efficacy as vaccine targets.

BACKGROUND: New candidate protective antigens for tick vaccine development may be identified by selecting and testing antigen candidates that play key biological functions. After blood-feeding, tick midgut overexpresses proteins that play essential functions in tick survival and disease transmission. Herein, Ornithodoros erraticus midgut transcriptomic and proteomic data were examined in order to select functionally significant antigens upregulated after feeding to be tested as vaccine candidate antigens. METHODS: Transcripts annotated as chitinases, tetraspanins, ribosomal protein P0 and secreted proteins/peptides were mined from the recently published O. erraticus midgut transcriptome and filtered in a second selection step using criteria based on upregulation after feeding, predicted antigenicity and expression in the midgut proteome. Five theoretical candidate antigens were selected, obtained as recombinant proteins and used to immunise rabbits: one chitinase (CHI), two tetraspanins (TSPs), the ribosomal protein P0 (RPP0) and one secreted protein PK-4 (PK4). RESULTS: Rabbit vaccination with individual recombinant candidates induced strong humoral responses that mainly reduced nymph moulting and female reproduction, providing 30.2% (CHI), 56% (TSPs), 57.5% (RPP0) and 57.8% (PK4) protection to O. erraticus infestations and 19.6% (CHI), 11.1% (TSPs), 0% (RPP0) and 8.1% (PK4) cross-protection to infestations by the African tick Ornithodoros moubata. The joint vaccine efficacy of the candidates was assessed in a second vaccine trial reaching 66.3% protection to O. erraticus and 25.6% cross-protection to O. moubata. CONCLUSIONS: These results (i) indicate that argasid chitinases and RPP0 are promising protective antigens, as has already been demonstrated for ixodid chitinases and RPP0, and could be included in vaccines targeting multiple tick species; (ii) reveal novel protective antigens tetraspanins and secreted protein PK-4, never tested before as protective antigens in ticks; and (iii) demonstrate that multi-antigenic vaccines increased vaccine efficacy compared with individual antigens. Lastly, our data emphasize the value of the tick midgut as a source of protective candidate antigens in argasids for tick control.

Therapeutic Vaccines Against Human Papilloma Viruses: Achievements and Prospects.

Human papillomaviruses of high carcinogenic risk (HR HPVs) are major etiological agents of malignant diseases of the cervix, vulva, penis, anal canal, larynx, head, and neck. Prophylactic vaccination against HPV, which mainly covers girls and women under 25, does not prevent vertical and horizontal HPV transmission in infants and children and does not have a therapeutic effect. As a result, a significant proportion of the population is not protected from the HPV infection and development of HPV-associated neoplastic transformation and cancer, which indicates the need for development and introduction of therapeutic HPV vaccines. Unlike prophylactic vaccines aimed at the formation of virus-neutralizing antibodies, therapeutic vaccines elicit cellular immune response leading to the elimination of infected and malignant cells expressing viral proteins. The ideal targets for vaccine immunotherapy are highly conserved HR HPV oncoproteins E6 and E7 expressed in precancerous and tumor tissues. Here, we describe expression of these proteins during different stages of HPV infection, their antigenic and immunogenic properties, and T-cell epitopes, the response to which correlates with natural regression of HPV-induced neoplastic changes. The review describes patterns of E6 and E7 oncoproteins presentation to the immune system as components of candidate vaccines along with the results of the most promising preclinical trials and animal models used in these trials. Special attention is paid to vaccine candidates which have shown efficacy in clinical trials in patients with HPV-associated neoplastic changes.

Antigenicity assessment of the Theileria equi merozoite antigen (EMA-2) expressed in Pichia pastoris in mice and horses.

Equine theileriosis is a severe equine disease caused by the protozoan Theileria equi, which is prevalent in tropical and subtropical areas. In this study, a recombinant equi merozoite antigen-2 (rEMA-2) of T. equi was used as an immunogen. Two groups of 10 mice each were divided into control and vaccinated groups. Sixty mares seronegative for theileriosis were divided in two groups, one vaccinated and another group as a control animal. Mice and mares of the vaccinated groups were inoculated with 150 µL of the vaccine containing 50 µg of rEMA-2 and 2 mL of the vaccine containing 200 µg of rEMA-2, respectively, at days 0 and 21. The immunogenicity of rEMA-2 was evaluated by ELISA and fluorescent antibody test (IFAT) using serum from vaccinated mice, mares and antigenicity in naturally infected horse. At every point throughout the ELISA study, there were significant differences between the vaccinated and control groups (p < 0.05). The vaccine induced 3- and 4-fold IgG increases in mice at the 14th and 28th day, respectively, compared to the control group. The horses' IgG dynamics showed a significant (p < 0.05) increase in the total IgG titer as early as day 7, which increased until day 28 at which time a more significant (p < 0.001) IgG titer was observed. In evaluating the isotypes, we observed a trend similar to that of total IgG, where IgG(T) (IgG3-5) were significantly (p < 0.05) more elevated than the other isotypes analyzed, followed by IgGb (IgG4-7) and IgGa (IgG1). Positive fluorescence was detected by IFAT, suggesting that the protein is immunogenic and conserves some epitopes identical to the native T. equi antigens present in the equine blood smear. Thus, our results suggest that rEMA-2 can be a promising vaccinal antigen.

Assessment of the immunogenicity of the leptospiral LipL32, LigAni, and LigBrep recombinant proteins in the sheep model.

Veterinary leptospirosis vaccines are composed of bacterins and present limitations, for example, the need for bacteriological culture and serovar-dependent immunity. Recombinant antigens represent a promising alternative. LigAni, LigBrep, and LipL32 proteins have been shown to promote a protective immune response against the homologous challenge in hamsters. Therefore, the next step is to evaluate the immunological properties of these immunogens in the actual hosts, as ruminants, which has never been performed before. The objective of this study was to evaluate the immunogenicity and potential adverse effects of the recombinant proteins LigAni, LigBrep, and LipL32 in the ovine model. For this, 16 Santa Inês sheep were allocated into three groups: two experimental (Groups A and B) and one control group (Group C). Group A was inoculated with a formulation containing the recombinant proteins in combination with the aluminum hydroxide adjuvant; Group B was inoculated with a formulation containing the recombinant proteins in combination with the Montanide adjuvant; and Group C was inoculated with adjuvants only. The results revealed that formulations containing the recombinant proteins induced total IgG seroconversion and led to a significant increase in antibody titers in the sheep model. Besides, there were no clinical changes or adverse effects. Thus, LigAni, LigBrep, and LipL32 proteins elicited a significant humoral immune response with elevated serum IgG levels, demonstrating that they possess the immunogenic and safety characteristics necessary to sustain their potential use as leptospirosis vaccines in the ruminant model.

Optimization of induction parameters, structure quality assessment by ATR-FTIR and in silico characterization of expressed recombinant polcalcin in three different strains of Escherichia coli.

The inhalation of Chenopodium album (C. album) pollen, especially polcalcin (Che a 3), has been reported as a significant source of allergic respiratory symptoms. This study was conducted to characterize biophysical differences of recombinant polcalcin come from three different Escherichia coli strains using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), optimize recombinant polcalcin expression, and linear B-cell epitopes identification using in silico methods. ATR-FTIR results of purified proteins showed spectra intensity variance in the amide I region, while there were no changes in pick position and shape of the bands. SHuffle® T7 Express was selected for subsequent optimization due to ability in the correction of the mis-oxidized bonds and promotes proper folding which was validated by ATR-FTIR analysis results. Then, Response Surface Methodology was performed to optimize critical factors including induction temperature, duration of induction, and concentration of inducer. The best partitioning conditions were achieved in 1.5 mM IPTG for 10.97 h at 29.9 °C. Finally, prediction of polcalcin B-cell epitopes was carried out which indicated the presence of 4 different epitopes. Together, the results may help to the development of diagnostic approaches and also vaccine manufacture for desensitization and modulation of the allergic response in patients.

Cloning and Expression of Mannheimia haemolytica PlpE Gene in Escherichia coli and its Immunogenicity Assessment.

Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the causative agents of shipping fever in cattle. Current vaccines against M. haemolytica are moderately efficacious since they do not provide complete protection against the disease. Production of an economic vaccine for protecting farm animals against M. haemolytica has attracted the attention of many scientists. The outer membrane proteins (OMPs) play a major role in the pathogenesis and immunogenicity of M. haemolytica. Research on M. haemolytica OMPs has shown that antibodies to a particular OMP may be important in immune protection. In the current study, the gene for M. haemolytica OMP PlpE was cloned into the expression vector pET26-b, and then expressed in Escherichia coli BL21. The expression of the protein was carried out by the induction of cultured Escherichiacoli Bl21 cells with 1mM isopropyl-&beta;-D-thiogalactopyranoside. The recombinant PlpE was purified using Ni-NTA agarose resin, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the expressed protein was analyzed by western blotting. It was revealed that rPlpE was expressed and produced properly. To assess the immunogenicity of the recombinant protein, the purified rPlpE was used as an antigen for antibody production in goats. The observations suggested that the produced recombinant protein can be used as a antigen for developing diagnostic tests and or as a vaccine candidate.

Screening and identification of a human domain antibody against Brucella abortus VirB5.

Brucellosis is caused by the genus Brucella. Brucella is widely distributed in cattle, swine, sheep, goat and other mammals including human. Animal brucellosis causes severe economic losses and affects related international transportation and trade. Human brucellosis causes both acute and chronic symptoms of multi-organ dysfunction. Brucella type IV secretion system (T4SS) VirB5 was required for macrophages infection and essential for virulence in mice. VirB5 is located on the cell surface and serves as a specific adhesin targeting host cell receptors. The aim of this study was to isolate and characterize a specific human domain antibody against Brucella abortus (B. abortus) VirB5 from human single domain antibody (sdAb or VHH) phage display library. Following five rounds of screening, an sdAb named as BaV5VH4 showed the highest affinity by enzyme-linked immunosorbent assay (ELISA). Its interaction with B. abortus VirB5 was verified by binding assay, dot blot and molecular docking. These findings in this paper could greatly help elucidate the molecular mechanisms of Brucella infection, and accelerate the development of sdAbs-based vaccines and neutralizing therapeutics of brucellosis.

Assessment of the impact of manufacturing changes on the physicochemical properties of the recombinant vaccine carrier ExoProtein A.

Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content.

Validation of Multiplex Serology detecting human herpesviruses 1-5.

Human herpesviruses (HHV) cause a variety of clinically relevant conditions upon primary infection of typically young and immunocompetent hosts. Both primary infection and reactivation after latency can lead to more severe disease, such as encephalitis, congenital defects and cancer. Infections with HHV are also associated with cardiovascular and neurodegenerative disease. However, most of the associations are based on retrospective case-control analyses and well-powered prospective cohort studies are needed for assessing temporality and causality. To enable comprehensive investigations of HHV-related disease etiology in large prospective population-based cohort studies, we developed HHV Multiplex Serology. This methodology represents a low-cost, high-throughput technology that allows simultaneous measurement of specific antibodies against five HHV species: Herpes simplex viruses 1 and 2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus. The newly developed HHV species-specific ('Monoplex') assays were validated against established gold-standard reference assays. The specificity and sensitivity of the HHV species-specific Monoplex Serology assays ranged from 92.3% to 100.0% (median 97.4%) and 91.8% to 98.7% (median 96.6%), respectively. Concordance with reference assays was very high with kappa values ranging from 0.86 to 0.96 (median kappa 0.93). Multiplexing the Monoplex Serology assays resulted in no loss of performance and allows simultaneous detection of antibodies against the 5 HHV species in a high-throughput manner.

Methodologies for data collection in congenital haemophilia with inhibitors (CHwI): critical assessment of the literature and lessons learned from recombinant factor VIIa.

AIMS: To systematically review the effectiveness of on-demand treatment with recombinant coagulation factor VIIa (rFVIIa) in congenital haemophilia with inhibitors and, if feasible, perform a meta-analysis of the data. MATERIALS AND METHODS: Publications from Embase® , MEDLINE® , MEDLINE® In-Process and the Cochrane Central Register of Controlled Trials were searched. Selected publications were reviewed for inclusion by two independent expert reviewers. Discrepancies were reconciled by a third independent reviewer. Data from selected studies were extracted using a predefined grid to ensure uniform and comparable results were captured. RESULTS: A systematic search (cut-off date of 2 May 2016) identified 20 studies (13 observational; seven randomized controlled trials). All studies were of sufficient quality to include in this analysis and comprised 1221 participants, with 5981 bleeds in 746 individuals treated with rFVIIa. Haemostatic overall effectiveness of the individual studies identified ranged from 68% to 100% at ≤12 hours, 86% to 96% at 13-24 hours and 76% to 99% at 24-48 hours with rFVIIa <100 µg/kg, with similar rates reported for the ≥250 µg/kg dose. However, heterogeneity between the studies precluded pooling of results. CONCLUSIONS: Data from the individual studies confirmed that rFVIIa is an effective therapy for the on-demand treatment of bleeds in congenital haemophilia with inhibitors. However, the high levels of heterogeneity between studies precluded pooling of results for a valid, reliable or precise summary measure. There remains a need to implement standardized clinical definitions and measurements for the effectiveness and safety of haemophilia therapies in future clinical trials.

The path to VICTORy - a beginner's guide to success using commercial research antibodies.

Commercial research antibodies are crucial tools in modern cell biology and biochemistry. In the USA some $2 billion a year are spent on them, but many are apparently not fit-for-purpose, and this may contribute to the 'reproducibility crisis' in biological sciences. Inadequate antibody validation and characterization, lack of user awareness, and occasional incompetence amongst suppliers have had immense scientific and personal costs. In this Opinion, I suggest some paths to make the use of these vital tools more successful. I have attempted to summarize and extend expert views from the literature to suggest that sustained routine efforts should made in: (1) the validation of antibodies, (2) their identification, (3) communication and controls, (4) the training of potential users, (5) the transparency of original equipment manufacturer (OEM) marketing agreements, and (5) in a more widespread use of recombinant antibodies (together denoted the 'VICTOR' approach).