Intensified high yield production of GMMA based vaccines for high burden neglected disease

Vaccines are among the most important public health interventions currently available. The global demand for vaccines is growing due to global population growth, ongoing global immunization campaigns and the increase of antibiotic-resistant bacteria. Vaccines are of high importance in Low- and Middle-Income Countries (LMIC) where millions of deaths occur annually due to vaccines-preventable diseases. Global coverage would need at least 200 million doses annually of a two-dose vaccine to cover the at-risk birth cohort, and considerably more for catch up vaccination during a roll out phase. Especially for vaccines against diseases in LMIC, there is a major added constraint: the vaccine production cost per unit and manufacturing facilities investments must be minimized to ensure the vaccines will be both viable and sustainable. For diseases caused by Gram-negative bacteria, the vaccine platform based on the GMMA technology is promising. GMMA are highly immunogenic outer membrane exosomes produced from genetically engineered bacteria modified for high yield hyper-blebbing that can be purified with minimal downstream processing. Simple robust scalable processes make GMMA ideal for cost-conscious vaccines. Please click Download on the upper right corner to see the full abstract

Analytical development to support manufacturing of a sustainable vaccine against Invasive Nontyphoidal Salmonellosis

GVGH is developing a candidate trivalent Salmonella vaccine to fight invasive nontyphoidal Salmonellosis (iNTS) and typhoid fever, especially aimed for sub-Saharan Africa to impact disease burden and to reduce anti-microbial resistance spread. This trivalent vaccine may be the only viable option for a sustainable iNTS vaccine in sub-Saharan Africa over the separate administration of Typhoid Conjugate Vaccines (TCV) and a vaccine against iNTS. GVGH generated the iNTS-TCV formulation by combining the GMMA technology for the iNTS components, S. Typhimurium (STm) and S. Enteritidis (SEn) GMMA adsorbed on Alhydrogel, and the Vi-CRM197 glycoconjugate, originally developed by GVGH and recently WHO prequalified as TCV TYPHIBEV by Biological E Ltd (Hyderabad, India). A set of analytical methods to support the vaccine lot release and characterization have been developed by GVGH. In particular, to quantify the key active ingredients of iNTS components a competitive ELISA-based method (FAcE, Formulated Alhydrogel competitive ELISA assay) has been setup and characterized in terms of specificity, accuracy and precision. Vi component is instead characterized by means of HPAEC-PAD method, able to specifically identify and quantify the total polysaccharide in the final drug product. With regard to safety assessment, a Monocyte Activation Test (MAT) has been developed as to monitor the intrinsic pyrogenicity of GMMA-based vaccines and applied as surveillance test for the Phase 1 clinical lot, with the plan to set release criteria based on clinical experience. In vivo potency assay has been set to characterize the immunogenicity of vaccine lots in comparison to freshly formulated material at the time of release and during real-time stability. A significant antibody response to each of the active ingredients of the trivalent vaccine is raised in mice and assessed by Parallel Line Assay. Overall, the applied analytical panel and the results support the development of an iNTS-TCV vaccine as a viable option for a sustainable iNTS vaccine in sub-Saharan Africa

Correction: An automated system for the objective evaluation of human gustatory sensitivity using tongue biopotential recordings.

[This corrects the article DOI: 10.1371/journal.pone.0177246.]

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

AbstractThe 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

Structural-functional characterization of the cathodic haemoglobin of the conger eel Conger conger: molecular modelling study of an additional phosphate-binding site.

The protein sequence data for the alpha- and beta-chains have been deposited in the SWISS-PROT and TrEMBL protein knowledgebase under the accession numbers P83479 and P83478 respectively. The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the alpha- and beta-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-alpha(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule