A flow cytometry-based assay to determine the ability of anti-Streptococcus pyogenes antibodies to mediate monocytic phagocytosis in human sera.

Streptococcus pyogenes, commonly referred to as Group A Streptococcus (Strep A), causes a spectrum of diseases, with the potential to progress into life-threatening illnesses and autoimmune complications. The escalating threat of antimicrobial resistance, stemming from the prevalent reliance on antibiotic therapies to manage Strep A infections, underscores the critical need for the development of disease control strategies centred around vaccination. Phagocytes play a critical role in controlling Strep A infections, and phagocytosis-replicating assays are essential for vaccine development. Traditionally, such assays have employed whole-blood killing or opsonophagocytic methods using HL-60 cells as neutrophil surrogates. However, assays mimicking Fcγ receptors- phagocytosis in clinical contexts are lacking. Therefore, here we introduce a flow cytometry-based method employing undifferentiated THP-1 cells as monocytic/macrophage model to swiftly evaluate the ability of human sera to induce phagocytosis of Strep A. We extensively characterize the assay's precision, linearity, and quantification limit, ensuring robustness. By testing human pooled serum, the assay proved to be suitable for the comparison of human sera's phagocytic capability against Strep A. This method offers a valuable complementary assay for clinical studies, addressing the gap in assessing FcγR-mediated phagocytosis. By facilitating efficient evaluation of Strep A -phagocyte interactions, it may contribute to elucidating the mechanisms required for the prevention of infections and inform the development of future vaccines and therapeutic advancements against Strep A infections.

O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

Putative correlates of protection against shigellosis assessing immunomarkers across responses to S. sonnei investigational vaccine.

Shigella spp. are a leading bacterial cause of diarrhea. No widely licensed vaccines are available and there is no generally accepted correlate of protection. We tested a S. sonnei Generalized Modules for Membrane Antigen (GMMA)-based vaccine (1790GAHB) in a phase 2b, placebo-controlled, randomized, controlled human infection model study (NCT03527173) enrolling healthy United States adults aged 18-50 years. We report analyses evaluating immune responses to vaccination, with the aim to identify correlates of risk for shigellosis among assessed immunomarkers. We found that 1790GAHB elicited S. sonnei lipopolysaccharide specific α4ß7+ immunoglobulin (Ig) G and IgA secreting B cells which are likely homing to the gut, indicating the ability to induce a mucosal in addition to a systemic response, despite parenteral delivery. We were unable to establish or confirm threshold levels that predict vaccine efficacy facilitating the evaluation of vaccine candidates. However, serum anti-lipopolysaccharide IgG and bactericidal activity were identified as potential correlates of risk for shigellosis.

Age-dependent acquisition of IgG antibodies to Shigella serotypes-a retrospective analysis of seroprevalence in Kenyan children with implications for infant vaccination.

Background: Shigellosis mainly affects children under 5 years of age living in low- and middle-income countries, who are the target population for vaccination. There are, however, limited data available to define the appropriate timing for vaccine administration in this age group. Information on antibody responses following natural infection, proxy for exposure, could help guide vaccination strategies. Methods: We undertook a retrospective analysis of antibodies to five of the most prevalent Shigella serotypes among children aged <5 years in Kenya. Serum samples from a cross-sectional serosurvey in three Kenyan sites (Nairobi, Siaya, and Kilifi) were analyzed by standardized ELISA to measure IgG against Shigella sonnei and Shigella flexneri 1b, 2a, 3a, and 6. We identified factors associated with seropositivity to each Shigella serotype, including seropositivity to other Shigella serotypes. Results: A total of 474 samples, one for each participant, were analyzed: Nairobi (n = 169), Siaya (n = 185), and Kilifi (n = 120). The median age of the participants was 13.4 months (IQR 7.0-35.6), and the male:female ratio was 1:1. Geometric mean concentrations (GMCs) for each serotype increased with age, mostly in the second year of life. The overall seroprevalence of IgG antibodies increased with age except for S. flexneri 6 which was high across all age subgroups. In the second year of life, there was a statistically significant increase of antibody GMCs against all five serotypes (p = 0.01-0.0001) and a significant increase of seroprevalence for S. flexneri 2a (p = 0.006), S. flexneri 3a (p = 0.006), and S. sonnei (p = 0.05) compared with the second part of the first year of life. Among all possible pairwise comparisons of antibody seropositivity, there was a significant association between S. flexneri 1b and 2a (OR = 6.75, 95% CI 3-14, p < 0.001) and between S. flexneri 1b and 3a (OR = 23.85, 95% CI 11-54, p < 0.001). Conclusion: Children living in low- and middle-income settings such as Kenya are exposed to Shigella infection starting from the first year of life and acquire serotype-specific antibodies against multiple serotypes. The data from this study suggest that Shigella vaccination should be targeted to infants, ideally at 6 or at least 9 months of age, to ensure children are protected in the second year of life when exposure significantly increases.

Development and characterization of a hemolysis inhibition assay to determine functionality of anti-Streptolysin O antibodies in human sera.

The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies.

Comparison of Shigella GMMA and glycoconjugate four-component formulations in animals.

Shigellosis is leading bacterial cause of diarrhea with high prevalence in children younger than 5 years in low- and middle-income countries, and increasing number of reports of Shigella cases associated to anti-microbial resistance. No vaccines against Shigella are still licensed, but different candidates based on the O-antigen portion of lipopolysaccharides are in clinic. Generalized Modules for Membrane Antigens (GMMA) have been proposed as an alternative delivery system for the O-antigen, and a 4-component vaccine candidate (altSonflex1-2-3), containing GMMA from S. sonnei and S. flexneri 1b, 2a and 3a is being tested in a phase 1/2 clinical trial, with the aim to elicit broad protection against the most prevalent Shigella serotypes. Here, the 4-component GMMA vaccine candidate has been compared to a more traditional glycoconjugate formulation for the ability to induce functional antibodies in mice and rabbits. In mice, in the absence of Alhydrogel, GMMA induce higher IgG antibodies than glycoconjugates and stronger bactericidal titers against all Shigella serotypes. In the presence of Alhydrogel, GMMA induce O-antigen specific IgG levels similar to traditional glycoconjugates, but with a broader range of IgG subclasses, resulting in stronger bactericidal activity. In rabbits, GMMA elicit higher functional antibodies than glycoconjugates against S. sonnei, and similar responses to S. flexneri 1b, 2a and 3a, independently from the presence of Alhydrogel. Different O-antigen based vaccines against Shigella are now in clinical stage and it will be of particular interest to understand how the preclinical findings in the different animal models translate in humans.

Testing a Recombinant Form of Tetanus Toxoid as a Carrier Protein for Glycoconjugate Vaccines.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM197), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM197 glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

A next-generation GMMA-based vaccine candidate to fight shigellosis.

Shigellosis is a leading cause of diarrheal disease in low-middle-income countries (LMICs). Effective vaccines will help to reduce the disease burden, exacerbated by increasing antibiotic resistance, in the most susceptible population represented by young children. A challenge for a broadly protective vaccine against shigellosis is to cover the most epidemiologically relevant serotypes among >50 Shigella serotypes circulating worldwide. The GMMA platform has been proposed as an innovative delivery system for Shigella O-antigens, and we have developed a 4-component vaccine against S. sonnei, S. flexneri 1b, 2a and 3a identified among the most prevalent Shigella serotypes in LMICs. Driven by the immunogenicity results obtained in clinic with a first-generation mono-component vaccine, a new S. sonnei GMMA construct was generated and combined with three S. flexneri GMMA in a 4-component Alhydrogel formulation (altSonflex1-2-3). This formulation was highly immunogenic, with no evidence of negative antigenic interference in mice and rabbits. The vaccine induced bactericidal antibodies also against heterologous Shigella strains carrying O-antigens different from those included in the vaccine. The Monocyte Activation Test used to evaluate the potential reactogenicity of the vaccine formulation revealed no differences compared to the S. sonnei mono-component vaccine, shown to be safe in several clinical trials in adults. A GLP toxicology study in rabbits confirmed that the vaccine was well tolerated. The preclinical study results support the clinical evaluation of altSonflex1-2-3 in healthy populations, and a phase 1-2 clinical trial is currently ongoing.

Study of Agonists of TLRs as Vaccine Adjuvants.

Vaccines adjuvants are critically needed to enhance the effectiveness of subunit vaccines. Due to their ability to link the innate with the adaptive immune response, Toll-like receptor (TLR) agonists have received great attention as adjuvants in vaccines against severe and complex diseases such as cancer, AIDS, and malaria. Here, we describe in vitro assays, e.g., the Monocyte Activation Test, TLR-specific activation assay, and TLR-blocking experiments, used to assess TLR agonists adjuvanted vaccines' safety and to characterize their ability to stimulate the innate immunity. Such assays are physiologically relevant as they work with human cells and allow to overcome the complexity and variability related to in vivo assays.

Characterization of Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-Antigens in Human Sera.

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies.

Functional assays to evaluate antibody-mediated responses against Shigella: a review.

Shigella is a major global pathogen and the etiological agent of shigellosis, a diarrheal disease that primarily affects low- and middle-income countries. Shigellosis is characterized by a complex, multistep pathogenesis during which bacteria use multiple invasion proteins to manipulate and invade the intestinal epithelium. Antibodies, especially against the O-antigen and some invasion proteins, play a protective role as titres against specific antigens inversely correlate with disease severity; however, the context of antibody action during pathogenesis remains to be elucidated, especially with Shigella being mostly an intracellular pathogen. In the absence of a correlate of protection, functional assays rebuilding salient moments of Shigella pathogenesis can improve our understanding of the role of protective antibodies in blocking infection and disease. In vitro assays are important tools to build correlates of protection. Only recently animal models to recapitulate human pathogenesis, often not in full, have been established. This review aims to discuss in vitro assays to evaluate the functionality of anti-Shigella antibodies in polyclonal sera in light of the multistep and multifaced Shigella infection process. Indeed, measurement of antibody level alone may limit the evaluation of full vaccine potential. Serum bactericidal assay (SBA), and other functional assays such as opsonophagocytic killing assays (OPKA), and adhesion/invasion inhibition assays (AIA), are instead physiologically relevant and may provide important information regarding the role played by these effector mechanisms in protective immunity. Ultimately, the review aims at providing scientists in the field with new points of view regarding the significance of functional assays of choice which may be more representative of immune-mediated protection mechanisms.

From an in vivo to an in vitro relative potency (IVRP) assay to fully characterize a multicomponent O-antigen based vaccine against Shigella.

Outer membrane vesicles (OMV) represent an innovative platform for the design of polysaccharide based vaccines. Generalized Modules for Membrane Antigens (GMMA), OMV released from engineered Gram-negative bacteria, have been proposed for the delivery of the O-Antigen, key target for protective immunity against several pathogens including Shigella. altSonflex1-2-3 is a GMMA based vaccine, including S. sonnei and S. flexneri 1b, 2a and 3a O-Antigens, with the aim to elicit broad protection against the most prevalent Shigella serotypes, especially affecting children in low-middle income countries. Here we developed an In Vitro Relative Potency assay, based on recognition of O-Antigen by functional monoclonal antibodies selected to bind the key epitopes of the different O-Antigen active ingredients, directly applied to our Alhydrogel-formulated vaccine. Heat-stressed altSonflex1-2-3 formulations were generated and extensively characterized. The impact of detected biochemical changes in in vivo and in vitro potency assays was assessed. The overall results showed how the in vitro assay can replace the use of animals, overcoming the inherently high variability of in vivo potency studies. The entire panel of physico-chemical methods developed will contribute to detect suboptimal batches and will be valuable to perform stability studies. The work on Shigella vaccine candidate can be easily extended to other O-Antigen based vaccines.

Streptococcus pyogenes Colonization in Children Aged 24-59 Months in the Gambia: Impact of Live Attenuated Influenza Vaccine and Associated Serological Responses.

BACKGROUND: Immunity to Streptococcus pyogenes in high burden settings is poorly understood. We explored S. pyogenes nasopharyngeal colonization after intranasal live attenuated influenza vaccine (LAIV) among Gambian children aged 24-59 months, and resulting serological response to 7 antigens. METHODS: A post hoc analysis was performed in 320 children randomized to receive LAIV at baseline (LAIV group) or not (control). S. pyogenes colonization was determined by quantitative polymerase chain reaction (qPCR) on nasopharyngeal swabs from baseline (day 0), day 7, and day 21. Anti-streptococcal IgG was quantified, including a subset with paired serum before/after S. pyogenes acquisition. RESULTS: The point prevalence of S. pyogenes colonization was 7%-13%. In children negative at day 0, S. pyogenes was detected at day 7 or 21 in 18% of LAIV group and 11% of control group participants (P = .12). The odds ratio (OR) for colonization over time was significantly increased in the LAIV group (day 21 vs day 0 OR, 3.18; P = .003) but not in the control group (OR, 0.86; P = .79). The highest IgG increases following asymptomatic colonization were seen for M1 and SpyCEP proteins. CONCLUSIONS: Asymptomatic S. pyogenes colonization appears modestly increased by LAIV, and may be immunologically significant. LAIV could be used to study influenza-S. pyogenes interactions. Clinical Trials Registration. NCT02972957.

Elucidating the role of N-acetylglucosamine in Group A Carbohydrate for the development of an effective glycoconjugate vaccine against Group A Streptococcus.

Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate against Group A Streptococcus infections. Native GAC consists of a polyrhamnose (polyRha) backbone with N-acetylglucosamine (GlcNAc) at every second rhamnose residue. Both native GAC and the polyRha backbone have been proposed as vaccine components. Here, chemical synthesis and glycoengineering were used to generate a panel of different length GAC and polyrhamnose fragments. Biochemical analyses were performed confirming that the epitope motif of GAC is composed of GlcNAc in the context of the polyrhamnose backbone. Conjugates from GAC isolated and purified from a bacterial strain and polyRha genetically expressed in E. coli and with similar molecular size to GAC were compared in different animal models. The GAC conjugate elicited higher anti-GAC IgG levels with stronger binding capacity to Group A Streptococcus strains than the polyRha one, both in mice and in rabbits. This work contributes to the development of a vaccine against Group A Streptococcus suggesting GAC as preferable saccharide antigen to include in the vaccine.

Exploring the Role of GMMA Components in the Immunogenicity of a 4-Valent Vaccine against Shigella.

Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

Development and Characterization of a Luminescence-Based High-Throughput Serum Bactericidal Assay (L-SBA) to Assess Bactericidal Activity of Human Sera against Nontyphoidal Salmonella.

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials.

Complement-mediated serum bactericidal activity of antibodies elicited by the Shigella sonnei GMMA vaccine in adults from a shigellosis-endemic country: Exploratory analysis of a Phase 2a randomized study.

Shigella is associated with a significant burden of disease worldwide among individuals of all ages and is the major cause of moderate and severe diarrhea in children under five years of age in low- and middle-income countries. Several candidate vaccines against Shigella species are currently under clinical development. The investigational 1790GAHB vaccine against Shigella sonnei is based on GMMA (Generalized Modules for Membrane Antigens) technology. The vaccine was well tolerated and induced high antibody levels in early-phase clinical trials in both Shigella-endemic and non-endemic settings. The present analysis assessed the bactericidal activity of antibodies induced by 1790GAHB in healthy Kenyan adults during a phase 2a, controlled, randomized study (NCT02676895). Participants received two doses of 1790GAHB 4 weeks apart containing either 1.5/25 µg or 6/100 µg O antigen/protein, or active comparator vaccines (Control). Serum bactericidal activity (SBA) against S. sonnei was assessed at pre-vaccination (D1), 28 days post-first dose (D29) and 28 days post-second dose (D57), using a luminescence-based assay. Most participants had SBA titers above the lower limit of quantification of the assay at D1. SBA geometric mean titers increased 3.4-fold in the 1.5/25 µg group and 6.3-fold in the 6/100 µg group by D29 and were maintained at D57. There was no increase in SBA geometric mean titers in the Control group. A strong correlation was observed between SBA titers and anti-S. sonnei lipopolysaccharide serum immunoglobulin G antibody concentrations (Pearson correlation coefficient = 0.918), indicating that SBA can effectively complement enzyme-linked immunosorbent assay data by indicating the functionality of 1790GAHB-induced antibodies.

Antigen presentation by Follicular Dendritic cells to cognate B cells is pivotal for Generalised Modules for Membrane Antigens (GMMA) immunogenicity.

GMMA has been proposed as a potent technology platform for the design of safe, effective and affordable vaccines. As GMMA are vesicles blebbing out of the outer membrane of Gram-negative bacteria, they contain lipopolysaccharides, lipoproteins and peptidoglycans that stimulate immune cells via Toll-like Receptors 4 (TLR4) or TLR2. Being basically nanoparticles, GMMA can be efficiently captured by Follicular Dendritic Cells (FDC) for antigen presentation to cognate B cells. GMMA have shown to be highly immunogenic in preclinical and clinical studies and the engagement of TLR4 and TLR2 or antigen presentation by FDC may have a prominent role in GMMA immunogenicity, which is well worth investigating. By using GMMA derived from Shigella sonnei and Salmonella Typhimurium, we show for the first time that the antigen presentation by FDC to cognate B cells plays a major role in the induction of an effective humoral immune response upon immunization with GMMA by using both models. The engagement of TLR4 is critical to elicit an optimal antibody production, but its effect on antibody functionality is dependent on GMMA type and is dispensable when immunizing with Alum adjuvant, whereas TLR2 does not have any role for GMMA immunogenicity. Our findings represent a substantial advancement of the knowledge on GMMA mode of action and shed a light on novel perspectives for the design of safer and more effective GMMA-based vaccines. ONE SENTENCE SUMMARY: The study demonstrated that the antigen presentation by FDC to cognate B cells plays a major role for GMMA immunogenicity.

Setup and Characterization of a High-Throughput Luminescence-Based Serum Bactericidal Assay (L-SBA) to Determine Functionality of Human Sera against Shigella flexneri.

Shigellosis represents a major public health problem worldwide. The morbidity of the disease, especially in children in developing countries, together with the increase of antimicrobial resistance make a vaccine against Shigella an urgent medical need. Several vaccines under development are targeting Shigella lipopolysaccharide (LPS), whose extreme diversity renders necessary the development of multivalent vaccines. Immunity against Shigella LPS can elicit antibodies capable of killing bacteria in a serotype-specific manner. Therefore, although a correlation of protection against shigellosis has not been established, demonstration of vaccine-elicited antibody bactericidal activity may provide one means of vaccine protection against Shigella. To facilitate Shigella vaccine development, we have set up a high-throughput serum bactericidal assay based on luminescence readout (L-SBA), which has been already used to determine the functionality of antibodies against S. sonnei in multiple clinical trials. Here we present the setup and intra-laboratory characterization of L-SBA against three epidemiologically relevant Shigella flexneri serotypes using human sera. We assessed the linearity, repeatability and reproducibility of the method, demonstrating high assay specificity to detect the activity of antibodies against each homologous strain without any heterologous aspecificity against species-related and non-species-related strains; this assay is ready to be used to determine bactericidal activity of clinical sera raised by multivalent vaccines and in sero-epidemiological studies.