Multiple Techniques for Size Determination of Generalized Modules for Membrane Antigens from Salmonella typhimurium and Salmonella enteritidis

Abstract

In the last years, outer membrane vesicles have attracted a lot of attention for the development of vaccines against bacterial pathogens. Extracellular vesicles can be obtained in high yields by genetic mutations, resulting in generalized modules for membrane antigens (GMMA). Methods to check the quality, consistency of production, and stability of GMMA vaccines are of fundamental importance. In this context, analytical methods for size distribution determination and verifying the integrity and possible aggregation of GMMA particles are strongly needed. Herein, GMMA particle size distribution has been evaluated by means of three different techniques. Dynamic light scattering (DLS), multiangle light scattering (MALS) coupled with high-performance liquid chromatography–size exclusion chromatography (SEC), and nanoparticle tracking analysis (NTA) have been compared to characterize GMMA from different mutants of Salmonella typhimurium and Salmonella enteritidis strains. We found that the presence of O-antigen chains on GMMA determined higher Z-average diameters by DLS compared to size estimation by MALS and that the hydrodynamic diameter increased with the number of O-antigen chains per GMMA particle. In the case of SEC-MALS, the size of the whole population better reflects the size of the most abundant particles, whereas DLS diameter is more influenced by the presence of larger particles in the sample. SEC-MALS and NTA are preferable to DLS for the analysis of bimodal samples, as they better distinguish populations of different size. MALS coupled to a size exclusion chromatography module also allows checking the purity of GMMA preparations, allowing determination of generally occurring contaminants such as soluble proteins and DNA. NTA permits real-time visualization with simultaneous tracking and counting of individual particles, but it is deeply dependent on the choice of data analysis parameters. All of the three techniques have provided complementary information leading to a more complete characterization of GMMA particles.

GMMA and Glycoconjugate Approaches Compared in Mice for the Development of a Vaccine against Shigella flexneri Serotype 6

“…On the contrary, following fermentation of the GMMA-producing bacterial strains, two simple tangential flow filtration steps allow us to purify high yields of GMMA [10,40]. GMMA are complex systems, but a large panel of analytical methods have been developed to allow their full characterization [27,28]. Many of these methods can be applied to GMMA from different pathogens, as verified here for S. flexneri 6.…”

“…All mutated GMMA had similar average particle size by HPLC-SEC MALLS, but not by DLS where the length of the sugar chains impacted the hydrodynamic diameter [27]. All GMMA were characterized by a similar number of lipid A molecules per GMMA protein (Table 5).…”

“…GMMA size was determined by dynamic light scattering (DLS) and high performance liquid chromatography-size exclusion chromatography/multiangle light scattering (HPLC-SEC/MALS) as previously described [27]. GMMA purity was assessed by HPLC-SEC analysis [28]; total protein content was estimated by micro BCA using bovine serum albumin (BSA) as a reference following the manufacturer's instructions (Thermo Scientific, Waltham, MA, USA); OAg sugar content was quantified by determination of methyl pentoses (6-deoxyhexoses) with Dische colorimetric method [29].…”

GMMA Is a Versatile Platform to Design Effective Multivalent Combination Vaccines

“…S . Typhimurium GMMA (obtained from isolate 1418 Δ tolR mutant strain), S. sonnei GMMA (obtained from Δ tolR Δ virG Δ htrB 53G mutant strain) and Neisseria meningitidis serogroup B (MenB) GMMA (produced from a Δ synX , Δ ctra , Δ gna33 , Δ lpxL1 Neisseria meningitidis mutant strain) were produced and characterized as previously described [ 4 , 19 ]. Plasmodium falciparum circumsporozoite protein (CSP) and P. falciparum Pfs25 recombinant proteins (42.5 and 18 kDa, respectively) were kindly provided by the Malaria Vaccine Initiative (PATH, Seattle, WA, USA) and the Laboratory of Malaria Immunology and Vaccinology (HHS/NIH/NIAID, Bathesda, MD, USA), respectively.…”

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella

“…GMMA were produced from S. Typhimurium 2189 Δ tolR and from S. Enteritidis 618 Δ tolR strains ( SI Appendix , Materials and Methods ) and purified and characterized as previously described ( 26 , 28 , 38 ). For synthesis of glycoconjugates, OAg were purified from the same S. Typhimurium 2189 and S. Enteritidis 618 wild-type bacteria ( 21 , 39 ) and characterized as previously described ( 15 , 39 ).…”