Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella

Rossi, Omar; Molesti, Eleonora; Saul, Allan; Giannelli, Carlo; Micoli, Francesca; Necchi, Francesca

doi:10.3390/ht9020014

Cited by 26 publications

(37 citation statements)

References 30 publications

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“…Results of the assay were expressed as the IC50: the reciprocal serum dilution that produced a 50% reduction of luminescence, which corresponds to 50% growth inhibition of the bacteria present in the assay [ 49 , 50 ]. GraphPad Prism 7 software (GraphPad Softare, San Diego, California) was used for curve fitting and IC50 determination.…”

Section: Methodsmentioning

confidence: 99%

Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation

Richardson

Ravenscroft

Arato³

et al. 2021

Vaccines

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The pathogenic bacterium Shigella is a leading cause of diarrheal disease and mortality, disproportionately affecting young children in low-income countries. The increasing prevalence of antibiotic resistance in Shigella necessitates an effective vaccine, for which the bacterial lipopolysaccharide O-antigen is the primary target. S. flexneri serotype 6 has been proposed as a multivalent vaccine component to ensure broad protection against Shigella. We have previously explored the conformations of S. flexneri O-antigens from serogroups Y, 2, 3, and 5 that share a common saccharide backbone (serotype Y). Here we consider serogroup 6, which is of particular interest because of an altered backbone repeat unit with non-stoichiometric O-acetylation, the antigenic and immunogenic importance of which have yet to be established. Our simulations show significant conformational changes in serogroup 6 relative to the serotype Y backbone. We further find that O-acetylation has little effect on conformation and hence may not be essential for the antigenicity of serotype 6. This is corroborated by an in vivo study in mice, using Generalized Modules for Membrane Antigens (GMMA) as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the S. flexneri serotype 6 O-antigen.

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Section: Methodsmentioning

confidence: 99%

Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation

Richardson

Ravenscroft

Arato³

et al. 2021

Vaccines

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“…shaker, and the luminescence signal was measured using a luminometer (Viktor). A 4-parameter non-linear regression was applied to raw luminescence for all the sera dilutions tested as previously described (45). The SBA titer is reported in IC50, defined as serum dilutions giving 50% inhibition of the ATP level in the negative control well.…”

Section: Animal Experimentsmentioning

confidence: 99%

SalmonellaParatyphi A Outer Membrane Vesicles Displaying Vi Polysaccharide as a Multivalent Vaccine against Enteric Fever

et al. 2021

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Typhoid and paratyphoid fever have a high incidence worldwide and coexist in many geographical areas, especially in Low-Middle Income Countries (LMIC) in South and South East Asia. There is extensive consensus on the urgent need for better and affordable vaccines against systemic Salmonella infections. Generalized Modules for Membrane Antigens (GMMA), outer membrane exosomes shed by Salmonella bacteria genetically manipulated to increase blebbing, resemble the bacterial surface where protective antigens are displayed in their native environment.Here we engineered S. Paratyphi A using the pDC5-viaB plasmid to generate GMMA displaying the heterologous S. Typhi Vi antigen together with the homologous O:2 O-Antigen. The presence of both Vi and O:2 was confirmed by flow cytometry on bacterial cells and their amount was quantified on the resulting vesicles through a panel of analytical methods. When tested in mice, such GMMA induced a strong antibody response against both Vi and O:2 and these antibodies were functional in a serum bactericidal assay. Our approach yielded a bivalent vaccine candidate able to induce immune responses against different Salmonella serovars which could benefit LMIC residents and travellers.

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“…Here we report bactericidal activity of sera collected from participants of the phase 1 and extension studies using a complement-mediated high-throughput luminescence-based SBA assay developed at the GSK Vaccines Institute for Global Health ( 39 , 40 ). We also assessed the impact of baseline ELISA antibody levels on SBA titer levels and correlation between ELISA IgG levels and SBA titers.…”

Section: Introductionmentioning

confidence: 99%

Antibodies Elicited by the Shigella sonnei GMMA Vaccine in Adults Trigger Complement-Mediated Serum Bactericidal Activity: Results From a Phase 1 Dose Escalation Trial Followed by a Booster Extension

et al. 2021

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Shigella is the second most deadly diarrheal disease among children under five years of age, after rotavirus, with high morbidity and mortality in developing countries. Currently, no vaccine is widely available, and the increasing levels of multidrug resistance make Shigella a high priority for vaccine development. The single-component candidate vaccine against Shigella sonnei (1790GAHB), developed using the GMMA technology, contains the O antigen (OAg) portion of lipopolysaccharide (LPS) as active moiety. The vaccine was well tolerated and immunogenic in early-phase clinical trials. In a phase 1 placebo-controlled dose escalation trial in France (NCT02017899), three doses of five different vaccine formulations (0.06/1, 0.3/5, 1.5/25, 3/50, 6/100 µg of OAg/protein) were administered to healthy adults. In the phase 1 extension trial (NCT03089879), conducted 2–3 years following the parent study, primed individuals who had undetectable antibody levels before the primary series received a 1790GAHB booster dose (1.5/25 µg OAg/protein). Controls were unprimed participants immunized with one 1790GAHB dose. The current analysis assessed the functionality of sera collected from both studies using a high-throughput luminescence-based serum bactericidal activity (SBA) assay optimized for testing human sera. Antibodies with complement-mediated bactericidal activity were detected in vaccinees but not in placebo recipients. SBA titers increased with OAg dose, with a persistent response up to six months after the primary vaccination with at least 1.5/25 µg of OAg/protein. The booster dose induced a strong increase of SBA titers in most primed participants. Correlation between SBA titers and anti-S. sonnei LPS serum immunoglobulin G levels was observed. Results suggest that GMMA is a promising OAg delivery system for the generation of functional antibody responses and persistent immunological memory.

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Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella

Cited by 26 publications

References 30 publications

Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation

Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation

SalmonellaParatyphi A Outer Membrane Vesicles Displaying Vi Polysaccharide as a Multivalent Vaccine against Enteric Fever

Antibodies Elicited by the Shigella sonnei GMMA Vaccine in Adults Trigger Complement-Mediated Serum Bactericidal Activity: Results From a Phase 1 Dose Escalation Trial Followed by a Booster Extension

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