Assessment of Tunable Resistive Pulse Sensing (TRPS) Technology for Particle Size Distribution in Vaccine Formulations - A Comparative Study with Dynamic Light Scattering.

PURPOSE: A comparative assessment was performed to evaluate the potential of particle sizing by an ensemble based conventional dynamic light scattering (DLS) technique and an emerging technology based on tunable resistive pulse sensing (TRPS) using particle by particle approach by evaluating three different types of vaccine formulations representing three case studies and showing the limitation of each technique, instrument variability, sensitivity, and the resolution in mixed population. METHODS: Three types of in-house vaccine formulations- a protein antigen, an outer membrane vesicle and viral particles were simultaneously evaluated by TRPS based Exoid and two DLS instruments-Zetatrac and Zetasizer for particle size distribution, aggregates, and resolution of polydisperse species. RESULTS: The data from first case study show the risk of possible size overestimation and size averaging in polydisperse samples in DLS measurements which can be addressed by the TRPS analysis. It also shows how TRPS may be utilized only to large size antigens due to its limited size range. The second case study highlights the difference in the sensitivities of two DLS instruments working on the same principle. The third case study show that how TRPS can better resolve the large aggregate species compare to DLS in polydisperse samples. CONCLUSION: This analysis shows that TRPS can be used as an orthogonal technique in addition to conventional DLS based methods for more precise and in-depth characterization. Both techniques are efficient in size characterization and produce comparable results, however the choice will depend on the type of formulation and size range to be evaluated.

Nanostructured cochleates: a multi-layered platform for cellular transportation of therapeutics.

Among lipid-based nanocarriers, multi-layered cochleates emerge as a novel delivery system because of prevention of oxidation of hydrophobic and hydrophilic drugs, enhancement in permeability, and reduction in dose of drugs. It also improves oral bioavailability and increases the safety of a drug by targeting at a specific site with less side effects. Nanostructured cochleates are used as a carrier for the delivery of water-insoluble or hydrophobic drugs of anticancer, antiviral and anti-inflammatory action. This review article focuses on different methods for preparation of cochleates, mechanism of formation of cochleates, mechanism of action like cochleate undergoes macrophagic endocytosis and release the drug into the systemic circulation by acting on membrane proteins, phospholipids, and receptors. Advanced methods such as calcium-substituted and ß-cyclodextrin-based cochleates, novel techniques include microfluidic and modified trapping method. Cochleates showed enhancement in oral bioavailability of amphotericin B, delivery of factor VII, oral mucosal vaccine adjuvant-delivery system, and delivery of volatile oil. In near future, cochleate will be one of the interesting delivery systems to overcome the stability and encapsulation efficiency issues associated with liposomes. The current limiting factors for commercial preparation of cochleates involve high cost of manufacturing, lack of standardization, and specialized equipments.

Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.

[From vaccine manufacturing to its availability in pharmacy]. / De la fabrication d'un vaccin à sa mise à disposition en pharmacie.

After a brief overview of vaccine industry and the regulatory requirements for biologics, the biological and pharmaceutical manufacturing of vaccine is presented. Vaccine production specificities are discussed. They show that, despite recent efforts and progress, continuously adapting vaccine supply to demand "at any time and in any place" remains a challenge, for reasons inherent in biological production, which is a production in tight flow, structurally delicate (control of the biological hazard), and weakly reactive.

Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody.

Targeting of specific receptors on antigen-presenting cells is an appealing prospect in the production of novel nanoparticulate vaccines. In particular, the targeting of vaccines to dendritic cell (DC) subsets has been shown in models to significantly improve the induction of immune responses. This paper describes the evaluation of natural ligands, mannan and chitosan, and monoclonal antibodies as targeting motifs to enhance uptake of PLGA nanoparticle carriers by bovine DCs. To assess enhancement of uptake after the addition of natural ligands a bovine monocyte derived DC (MoDC) model was used. For the assessment of monoclonal antibody targeting, the model was expanded to include afferent lymph DCs (ALDCs) in a competitive uptake assay. Mannan, proved unsuccessful at enhancing uptake or targeting by MoDCs. Chitosan coated particle uptake could be impeded by the addition of mannan suggesting uptake may be mediated through sugar receptors. Inclusion of monoclonal antibodies specific for the DEC-205 (CD205) receptor increased the number of receptor expressing DCs associated with particles as well as the number of particles taken up by individual cells. These results support the further evaluation of active targeting of nanovaccines to DCs to enhance their immunogenicity in cattle and other large mammalian species including humans.

Sociological Environmental Causes are Insufficient to Explain Autism Changepoints of Incidence.

The Environmental Protection Agency (EPA) recently published a study analyzing time trends in the cumulative incidence of autistic disorder (AD) in the U.S., Denmark, and worldwide. A birth year changepoint (CP) around 1988 was identified. It has been argued that the epidemic rise in autism over the past three decades is partly due to a combination of sociologic factors along with the potential contribution of thimerosal containing vaccines. Our work conducted an expanded analysis of AD changepoints in CA and U.S., and determined whether changepoints in time trends of AD rates temporally coincide with changepoints for the proposed causative sociologic and environmental factors. Birth year changepoints were identified for 1980.9 [95% CI, 1978.6-1983.1], 1988.4 [95% CI, 1987.8-1989.0] and 1995.6 [95% CI, 1994.6-1996.6] for CA and U.S. data, confirming and expanding the EPA results. AD birth year changepoints significantly precede the changepoints calculated for indicators of increased social awareness of AD. Furthermore, the 1981 and 1996 AD birth year changepoints don't coincide with any predicted changepoints based on altered thimerosal content in vaccines nor on revised editions of the Diagnostic and Statistical Manual of Mental Disorders (DSM).

Simplified low-cost production of O-antigen from Salmonella Typhimurium Generalized Modules for Membrane Antigens (GMMA).

The Novartis Vaccines Institute for Global Health is developing vaccines using outer membrane particles, known as Generalized Modules for Membrane Antigens (GMMA). These are blebs of outer membrane and periplasm, shed from the surface of living Gram-negative bacteria following the targeted deletion of proteins involved in maintaining the integrity of the inner and outer membranes. The current study investigates the use of GMMA as starting material for extraction of membrane components, focusing on the O-antigen polysaccharide portion of lipopolysaccharide from Salmonella Typhimurium. We show that the amount of O-antigen extracted from GMMA by acid hydrolysis is comparable to the quantity extracted from whole wild type bacteria, but with less protein and DNA contaminants. Compared to conventional purification, GMMA enabled a reduction in the number of purification steps required to obtain the O-antigen polysaccharide with the same purity. Purification processes from GMMA and bacteria were characterised by similar final yields. Use of GMMA as starting material provides the possibility to simplify the purification process of O-antigen, with a consequent decrease in manufacturing costs of O-antigen-based glyconjugate vaccines against Salmonella strains and potentially other Gram-negative bacteria.

Desirable attributes of vaccines for deployment in low-resource settings.

A number of product development partnerships are actively developing new vaccines to combat infectious diseases in developing countries. To be effective, the products under development should not only be safe, efficacious, and affordable, but they should also have additional desirable technical attributes, including enhanced stability, efficient packaging, and improved ease of delivery. New technologies are now available to achieve these attributes; however, many of the technologies have yet to be adopted by the vaccine industry. This commentary discusses the opportunities and challenges associated with advancing such attributes, especially vaccine thermostability and dose sparing strategies, and the critical issues that must be addressed to bridge the gap between technology development and product development.

Developing plant-based vaccines against neglected tropical diseases: where are we?

Neglected tropical diseases (NTDs) impair the lives of 1 billion people worldwide, and threaten the health of millions more. Although vaccine candidates have been proposed to prevent some NTDs, no vaccine is available at the market yet. Vaccines against NTDs should be low-cost and needle-free to reduce the logistic cost of their administration. Plant-based vaccines meet both requirements: plant systems allow antigen production at low cost, and also yield an optimal delivery vehicle that prevents or delays digestive hydrolysis of vaccine antigens. This review covers recent reports on the development of plant-based vaccines against NTDs. Efforts conducted by a number of research groups to develop vaccines as a mean to fight rabies, cysticercosis, dengue, and helminthiasis are emphasized. Future perspectives are identified, such as the need to develop vaccination models for more than ten pathologies through a plant-based biotechnological approach. Current limitations on the method are also noted, and molecular approaches that might allow us to address such limitations are discussed.

Molecular characterization of thyroid hormone receptor beta from Schistosoma japonicum and assessment of its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice.

BACKGROUND: Thyroid hormones (TH) modulate growth, development and differentiation and metabolic processes by interacting with thyroid hormone receptors (THRs). The purpose of this study was to identify a novel thyroid hormone receptor beta encoding gene of Schistosoma japonicum (SjTHRß) and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice. METHODS: The full-length cDNA sequence of SjTHRß, its gene organization, and its transcript levels were characterized, and the phylogenetic relationship between THR, RAR and RXR from other organisms were analysis, the ability of this protein binding to a conserved DNA core motif, and its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice were evaluated. RESULTS: The SjTHRß cDNA was cloned, verified by 5' and 3' Rapid Amplification of cDNA Ends and shown to be polyadenylated at the 3'end, suggesting the transcript is full-length. SjTHRß is homologous to THRs from other species and has a predicted conservative DNA binding domain and ligand binding domain that normally characterizes these receptors. A comparative quantitative PCR analysis showed that SjTHRß was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHRß-DBD) and ligand binding domain (SjTHRß-LBD) were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHRß (rSjTHRß). Western blotting revealed that anti-rSjTHRß-LBD serum recognized two protein bands in extracts from 21 d worm with molecular sizes of approximately 95 kDa and 72 kDa. Electrophoretic mobility shift assay (EMSA) analysis showed that rSjTHRß-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHRß-LBD could induce partial protective efficacy(27.52% worm reduction and 29.50% liver eggs reduction)against schistosome infection. Enzyme-linked immunosorbent assay showed that mice vaccinated with recombinant SjTHRß-LBD (rSjTHRß-LBD) generated increased levels of specific IgG, IgG1 and IgG2a antibody. Bio-plex analysis demonstrated that rSjTHRß-LBD induced considerably higher levels of T helper 1 cytokines (IL-2, IL-12 and TNF-α) than T helper 2 cytokines (IL-10, IL-4), suggesting that rSjTHRß-LBD vaccination could stimulate mixed Th1/Th2 types with Th1 dominant immune responses. CONCLUSIONS: Our study presented here identified SjTHRß as a new schistosome THR that might play an important role in host-parasite interaction and be a vaccine candidate for schistosomiasis.

The Vaccine Formulation Laboratory: a platform for access to adjuvants.

Adjuvants are increasingly used by the vaccine research and development community, particularly for their ability to enhance immune responses and for their dose-sparing properties. However, they are not readily available to the majority of public sector vaccine research groups, and even those with access to suitable adjuvants may still fail in the development of their vaccines because of lack of knowledge on how to correctly formulate the adjuvants. This shortcoming led the World Health Organization to advocate for the establishment of the Vaccine Formulation Laboratory at the University of Lausanne, Switzerland. The primary mission of the laboratory is to transfer adjuvants and formulation technology free of intellectual property rights to academic institutions, small biotechnology companies and developing countries vaccine manufacturers. In this context, the transfer of an oil-in-water emulsion to Bio Farma, an Indonesian vaccine manufacturer, was initiated to increase domestic pandemic influenza vaccine production capacity as part of the national pandemic influenza preparedness plan.

Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization.

Dry-coated microprojections can deliver vaccine to abundant antigen-presenting cells in the skin and induce efficient immune responses and the dry-coated vaccines are expected to be thermostable at elevated temperatures. In this paper, we show that we have dramatically improved our previously reported gas-jet drying coating method and greatly increased the delivery efficiency of coating from patch to skin to from 6.5% to 32.5%, by both varying the coating parameters and removing the patch edge. Combined with our previous dose sparing report of influenza vaccine delivery in a mouse model, the results show that we now achieve equivalent protective immune responses as intramuscular injection (with the needle and syringe), but with only 1/30th of the actual dose. We also show that influenza vaccine coated microprojection patches are stable for at least 6 months at 23°C, inducing comparable immunogenicity with freshly coated patches. The dry-coated microprojection patches thus have key and unique attributes in ultimately meeting the medical need in certain low-resource regions with low vaccine affordability and difficulty in maintaining "cold-chain" for vaccine storage and transport.

Re-formulating drugs and vaccines for intranasal delivery: maximum benefits for minimum risks?

The challenges being faced by the pharmaceutical industry in terms of patent expiries and a sparse pipeline of new products are well documented, as are the risks and costs associated with developing new molecular entities. Major pharmaceutical companies are increasingly looking to augment their traditional core expertise in the discovery of small molecules with the development of biologicals (e.g. peptide-based, protein-based, antibody-based and nucleic-acid-based therapies), which are seen as a key element in achieving long-term growth. There is also considerable current interest in vaccines, both in the traditional area of mass immunization against infections and as a novel approach to disease treatment.

Evaluation of MHC-II peptide binding prediction servers: applications for vaccine research.

BACKGROUND: Initiation and regulation of immune responses in humans involves recognition of peptides presented by human leukocyte antigen class II (HLA-II) molecules. These peptides (HLA-II T-cell epitopes) are increasingly important as research targets for the development of vaccines and immunotherapies. HLA-II peptide binding studies involve multiple overlapping peptides spanning individual antigens, as well as complete viral proteomes. Antigen variation in pathogens and tumor antigens, and extensive polymorphism of HLA molecules increase the number of targets for screening studies. Experimental screening methods are expensive and time consuming and reagents are not readily available for many of the HLA class II molecules. Computational prediction methods complement experimental studies, minimize the number of validation experiments, and significantly speed up the epitope mapping process. We collected test data from four independent studies that involved 721 peptide binding assays. Full overlapping studies of four antigens identified binding affinity of 103 peptides to seven common HLA-DR molecules (DRB1*0101, 0301, 0401, 0701, 1101, 1301, and 1501). We used these data to analyze performance of 21 HLA-II binding prediction servers accessible through the WWW. RESULTS: Because not all servers have predictors for all tested HLA-II molecules, we assessed a total of 113 predictors. The length of test peptides ranged from 15 to 19 amino acids. We tried three prediction strategies - the best 9-mer within the longer peptide, the average of best three 9-mer predictions, and the average of all 9-mer predictions within the longer peptide. The best strategy was the identification of a single best 9-mer within the longer peptide. Overall, measured by the receiver operating characteristic method (AROC), 17 predictors showed good (AROC > 0.8), 41 showed marginal (AROC > 0.7), and 55 showed poor performance (AROC < 0.7). Good performance predictors included HLA-DRB1*0101 (seven), 1101 (six), 0401 (three), and 0701 (one). The best individual predictor was NETMHCIIPAN, closely followed by PROPRED, IEDB (Consensus), and MULTIPRED (SVM). None of the individual predictors was shown to be suitable for prediction of promiscuous peptides. Current predictive capabilities allow prediction of only 50% of actual T-cell epitopes using practical thresholds. CONCLUSION: The available HLA-II servers do not match prediction capabilities of HLA-I predictors. Currently available HLA-II prediction servers offer only a limited prediction accuracy and the development of improved predictors is needed for large-scale studies, such as proteome-wide epitope mapping. The requirements for accuracy of HLA-II binding predictions are stringent because of the substantial effect of false positives.

An economic evaluation of thermostable vaccines in Cambodia, Ghana and Bangladesh.

This paper evaluates the incremental health and programmatic cost impacts of new vaccine products, as compared to the standard vaccine products in multi-dose vials in Cambodia, Ghana, and Bangladesh. The authors use a cost-effectiveness model to estimate the impacts of introducing four thermostable vaccines with single-dose presentations: measles, yellow fever, bacille Calmette-Guérin, and diphtheria-tetanus-pertussis-hepatitis B. The effectiveness of all of the vaccines increases with the thermostable formats. The incremental costs associated with the introduction of thermostable vaccines increases for three out of four vaccines. Single-dose presentations of thermostable vaccines are potentially cost-effective interventions to reduce childhood deaths and disability in low-resource settings in Asia and Africa.