Characterization of O-antigen delivered by Generalized Modules for Membrane Antigens (GMMA) vaccine candidates against nontyphoidal Salmonella

Benedetto, Gianluigi De; Alfini, Renzo; Cescutti, Paola; Caboni, Mariaelena; Lanzilao, Luisa; Necchi, Francesca; Saul, Allan; MacLennan, Calman A.; Rondini, Simona; Micoli, Francesca

doi:10.1016/j.vaccine.2016.11.089

Cited by 70 publications

(98 citation statements)

References 44 publications

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“…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]. The amount of lipid A molecules in GMMA was assumed equal to lipopolysaccharide (LPS) core reducing end 2-keto-3-deoxy-octonate (KDO) and quantified by semicarbazide/HPLC-SEC method after sugar extraction [30].…”

Section: Gmma Characterizationmentioning

confidence: 99%

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

et al. 2020

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Shigella infections are one of the top causes of diarrhea throughout the world, with Shigella flexneri being predominant in developing countries. Currently, no vaccines are widely available and increasing levels of multidrug-resistance make Shigella a high priority for vaccine development. The serotype-specific O-antigen moiety of Shigella lipopolysaccharide has been recognized as a key target for protective immunity, and many O-antigen based candidate vaccines are in development. Recently, the Generalized Modules for Membrane Antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Here, these two technologies are compared for a vaccine against S. flexneri serotype 6. Genetic strategies for GMMA production, conjugation approaches for linkage of the O-antigen to CRM197 carrier protein, and a large panel of analytical methods for full vaccine characterization have been put in place. In a head-to-head immunogenicity study in mice, GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel. When formulated on Alhydrogel, GMMA and glycoconjugate elicited similar levels of persistent anti-O-antigen IgG with bactericidal activity. Glycoconjugates are a well-established bacterial vaccine approach, but can be costly, particularly when multicomponent preparations are required. With similar immunogenicity and a simpler manufacturing process, GMMA are a promising strategy for the development of a vaccine against Shigella.

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Section: Gmma Characterizationmentioning

confidence: 99%

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

et al. 2020

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“…GMMA contain pathogen‐associated molecular patterns, including toll‐like receptor ligands, which can act as self‐adjuvants in the immune responses they elicit . GMMA are highly immunogenic, effective, have low reactogenicity, are safe, and can be manufactured at low cost in a scalable process . The main limitation of GMMA is that it cannot perform human‐like post‐translational modifications (e.g., glycosylation).…”

Section: Outer Membrane Vesicle Vaccines Generalized Modules For Memmentioning

confidence: 99%

“…For this, in Shigella sonnei the tolR, galU , and msbB1 genes were replaced by antibiotic selection markers . For GMMA vaccine production in Salmonella , the tolR, msbB, htrB , and pagP genes can be deleted and replaced by antibiotic selection markers . The obtained bacterial strains are stored in a two‐tiered cell bank and then expanded for production.…”

Section: Outer Membrane Vesicle Vaccines Generalized Modules For Memmentioning

confidence: 99%

Emerging Technologies for Low‐Cost, Rapid Vaccine Manufacture

Kis

Shattock

Shah

et al. 2018

Biotechnology Journal

117

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To stop the spread of future epidemics and meet infant vaccination demands in low‐ and middle‐income countries, flexible, rapid and low‐cost vaccine development and manufacturing technologies are required. Vaccine development platform technologies that can produce a wide range of vaccines are emerging, including: a) humanized, high‐yield yeast recombinant protein vaccines; b) insect cell‐baculovirus ADDomer vaccines; c) Generalized Modules for Membrane Antigens (GMMA) vaccines; d) RNA vaccines. Herein, existing and future platforms are assessed in terms of addressing challenges of scale, cost, and responsiveness. To assess the risk and feasibility of the four emerging platforms, the following six metrics are applied: 1) technology readiness; 2) technological complexity; 3) ease of scale‐up; 4) flexibility for the manufacturing of a wide range of vaccines; 5) thermostability of the vaccine product at tropical ambient temperatures; and 6) speed of response from threat identification to vaccine deployment. The assessment indicated that technologies in the order of increasing feasibility and decreasing risk are the yeast platform, ADDomer platform, followed by RNA and GMMA platforms. The comparative strengths and weaknesses of each technology are discussed in detail, illustrating the associated development and manufacturing needs and priorities.

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“…LPS was extracted from S. sonnei by hot phenol extraction as previously reported (10), whereas OAg was extracted from S. flexneri 1b, 2a 3a and from Salmonella Typhimurium by direct acid hydrolysis as previously reported (14). All extracted polysaccharides were fully characterised in terms of sugar content, protein and nuclei acid impurities by a combination of analytical techniques, including High-Performance Liquid Size Exclusion Chromatography (31), micro BCA and absorption at 260 nm as previously reported (14).…”

Section: Methodsmentioning

confidence: 99%

“…Result of the assay is the IC50, the dilution of sera able to kill half of the bacteria present in the assay, thus representing the SBA titer of the sera. We have already demonstrated the possibility to use the L-SBA to determine the bactericidal activity of sera raised against S. sonnei GMMA in pre-clinical models (14). Here we present the further development of this method, showing its full characterisation using human sera, and in particular sera raised against S. sonnei GMMA based vaccine (1790GAHB) as model.…”

Section: Introductionmentioning

confidence: 99%

Intralaboratory evaluation of luminescence based high-throughput Serum Bactericidal Assay (L-SBA) to determine bactericidal activity of human sera againstShigella

Rossi

Molesti

Saul

et al. 2020

Preprint

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17Despite the huge decrease in deaths caused by Shigella worldwide in the last decades, shigellosis is 18 still causing over 200,000 deaths every year. No vaccine is currently available, and the morbidity of 19 disease coupled with the rise of antimicrobial resistance renders the introduction of an effective 20 vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not 21 been established yet, the demonstration of bactericidal activity of antibodies induced upon 22 vaccination may provide one means of functionality of antibodies induced on protecting against 23 Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-24 induced antibodies is the Serum Bactericidal Assay (SBA). 25Here we present the development and intra-laboratory characterisation of a high-throughput 26 luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving 27 bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high 28 specificity of the assay against homologous strain without any heterologous aspecificity detected 29 against species-related and not species-related strains. We assessed linearity, repeatability and 30 reproducibility of L-SBA on human sera. 31This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The 32 method has the potential of being applicable with similar performances to determine bactericidal 33 activity of any non-clinical and clinical sera that rely on complement mediated killing. 34 35 IMPORTANCE 36Shigella is an important cause of diarrhoea worldwide and antimicrobial resistance is on rise, thus 37 efforts by several groups are ongoing to produce a safe and effective vaccine against shigellosis. 38Although a clear immune correlate of protection has not been established, demonstration of 39 bactericidal capacity of sera from patients immunised with Shigella vaccines may provide one means 40 characterisation L-SBA on human sera of protecting against shigellosis. We have developed and fully characterised a novel high-throughput 41 L-SBA method for evaluation of functionality of antibodies raised against S. sonnei in human sera. 42This work will allow the clinical testing of human sera raised against GMMA-based and potentially 43 all vaccines producing antibodies than can work via complement mediated manner. 44 45 46 47 48 Diarrheal diseases, such as shigelloses and salmonelloses, are the second leading cause of death 49 worldwide, resulting in millions of deaths per year, mostly in developing countries (1). Shigella is 50 major cause of sustained endemic bacterial diarrhoea, especially in low and middle-income countries 51 where accessibility to clean water is restricted. Although the improvement of hygienic conditions in 52 the last decade has dramatically reduced the burden of the disease, Shigella is still responsible for 53 more than 200,000 deaths, with a third of them being in young children (1). On top of deaths...

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Characterization of O-antigen delivered by Generalized Modules for Membrane Antigens (GMMA) vaccine candidates against nontyphoidal Salmonella

Cited by 70 publications

References 44 publications

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

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

Emerging Technologies for Low‐Cost, Rapid Vaccine Manufacture

Intralaboratory evaluation of luminescence based high-throughput Serum Bactericidal Assay (L-SBA) to determine bactericidal activity of human sera againstShigella

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