Cost effective manufacturing methods for Public Health vaccines against Gram negative bacteria
Author(s)
Meloni, Eleonora
Date Issued
June 10, 2013
Type
Doctoral Thesis
Abstract
Infectious diseases, particularly in less developed countries, remain among the leading causes of global mortality, most of these infections are caused by Gram-negative bacteria which are the major pathogens responsible of neonatal sepsis and cause of a range of diarrheal, respiratory and invasive diseases.
The most frequent Gram-negative isolates are E.coli, Salmonella spp and Shigella spp.
Salmonella infection represents a considerable burden in both developing and developed countries. Efforts to reduce transmission of salmonellae by food and other routes must be implemented on a global scale thus the development of cost effective, needle-independent vaccines is a high priority.
Vaccine for Developing Countries should be designed to be inexpensive (less than one euro for association like UNICEF and GAVI), stable at room temperature, simple to administer to infants, readily administered by nonprofessionals using a needle-free method and the vaccines formulation would protect against multiple agents.
Novartis Vaccines Institute for Global Health (NVGH) aims to develop a broadly protective vaccine using outer membrane particles. These particles, called Generalized Modules for Membrane Antigens (GMMA), also known as outer membrane vesicles (OMV), are naturally shed from the surface of living Gram‐negative bacteria. GMMA are different from the detergent‐extracted OMV which are depleted of lipooligosaccharides and lipoproteins.
The purpose of this PhD project was to investigate the possibility to develop low cost vaccines applicable to Gram-negative strains, intended for neglected infectious diseases of impoverished communities, using the GMMA technology platform.
This work showed the possibility to use GMMA particles as starting material for membrane components extraction, optimizing new and innovative platform, particularly for LPS or proteins recovery, in order to decrease the costs of goods (COGs).
The present work is focused on the O: 4,5-antigen polysaccharide extraction (using the acid hydrolysis approach), purification and characterization from Salmonella Typhimurium SL1344 tolR- GMMA.
NVGH, to over produce and collect GMMA, is developing new methods using over producing GMMA mutant strain (the tolR gene deletion results in substantial overproduction of outer
membrane particles) and two consecutive TFF steps (Tangential Flow Filtration steps: micro-filtration at 0.2 μm and thereafter a second micro-filtration at 0.1 μm) at the end of the fermentation process to collect GMMA from bacterial suspensions.
NVGH has developed another innovative platform for O-antigen extraction from biomass at the end of the fermentation process, using the acid hydrolysis approach of whole bacterial culture.
The present work shows for the first time the possibility to use GMMA particles as O-antigens source (and in general as source of membrane components) due their membrane composition enriched in LPS. We have proven that, O-antigens can be extracted from GMMA using the direct acid hydrolysis method thus the consequent O-antigen purification process is easy and cost effective.
This work provides the basis for the development of new vaccines, glyconiugate or proteins based, exploiting the same approach used and developed for S. Tyhphimurium SL1344 tolR- GMMA, which could be applied to other Gram negative strains.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
Subjects
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