Search Results (11)

Salmonellosis remains a major cause of morbidity and mortality in low- and middle-income countries, despite the availability of effective vaccines against Salmonella enterica serovar Typhi (S. Typhi). In response, substantial efforts have been underway to develop vaccines against the key serovars responsible for invasive non-typhoidal Salmonella (iNTS) disease, such as S. Typhimurium and S. Enteritidis, as well as against S. Paratyphi A, which, together with S. Typhi, is responsible for enteric fever. The O-antigens (OAg) are considered potential protective antigens; therefore, the most advanced vaccine candidates focus on these moieties. However, no correlate of protection has been identified for either iNTS or paratyphoid fever, highlighting the importance of developing robust functional assays to assess vaccine-induced immunogenicity. In this study, we present the characterization of a high-throughput luminescence-based serum bactericidal assay (L-SBA) against multiple S. enterica serovars, using human sera. The assay was evaluated for repeatability, intermediate precision, linearity, and specificity against a panel of Salmonella strains belonging to serogroups O:4, O:9, and O:2, which were selected for their epidemiological relevance and diversity in OAg expression, quantity, and glucosylation/acetylation patterns. This assay will enable testing of clinical sera from vaccine trials to evaluate the breadth of the functional activity stimulated by current Salmonella vaccine candidates. L-SBA demonstrated an acceptable performance with all the tested strains, resulting in being linear, specific, and precise. This study also provided preliminary evidence that human sera containing antibodies against serogroup-specific OAg can efficiently kill Salmonella strains expressing OAg of the matched serovar, even in the presence of variation in OAg molecular weight, glucosylation, and acetylation. The L-SBA will enable testing of clinical sera from vaccine trials to evaluate the breadth of the functional activity stimulated by current Salmonella vaccine candidates. Full article

Background: Typhoid and paratyphoid fevers represent a global health burden, especially in Southern Asia, exacerbated by the increase in antimicrobial resistance. While vaccines against Salmonella Typhi have been successfully introduced, a vaccine against S. Paratyphi A is not available, yet. Efforts to develop an effective vaccine targeting both Salmonella serovars are currently ongoing. GVGH is developing a bivalent vaccine constituted by the Vi-CRM₁₉₇ typhoid conjugate vaccine (TCV), and the Salmonella Paratyphi A O-antigen (O:2), also conjugated to the CRM₁₉₇ carrier protein (O:2-CRM₁₉₇). In this work we have characterized a panel of S. Paratyphi A clinical isolates from endemic regions, differing in terms of their O:2 structural features. Methods: Rabbits were immunized with the S. Paratyphi A component of the vaccine candidate and the resulting sera were tested for their ability to bind and kill the isolates using flow cytometry and luminescence-based serum bactericidal assay (L-SBA). Results: The O:2-CRM₁₉₇ glycoconjugate induced a functional immune response in rabbits, effectively binding and killing a diverse panel of clinical isolates. The sera demonstrated bactericidal activity independent of the O:2 structural variations, including differences in O-acetylation and glucosylation levels. Additionally, the study found that the O:2-CRM₁₉₇ conjugate’s adsorption to Alhydrogel did not significantly impact its immunogenicity or bactericidal efficacy. Conclusions: The O:2-CRM₁₉₇ component of the bivalent vaccine candidate shows promise in providing broad protection against S. Paratyphi A isolates, regardless of their O-antigen structural variations. The ongoing clinical studies on human sera are expected to confirm these results. Full article

Shigellosis represents a significant global health concern particularly affecting children under 5 years in low- and middle-income countries (LMICs) and is associated with stunting and antimicrobial resistance. There is a critical need for an effective vaccine offering broad protection against the different Shigella serotypes. A correlate of protection has not yet been established but there is a general consensus about the relevant role of anti-O-Antigen-specific IgG and its functionality evaluated by the Serum Bactericidal Assay (SBA). This study aims to characterize a high-throughput luminescence-based SBA (L-SBA) against seven widespread Shigella serotypes. The assay was previously developed and characterized for S. sonnei and S. flexneri 1b, 2a, and 3a and has now been refined and extended to an additional five serotypes (S. flexneri 4a, 5b, 6, X, and Y). The characterization of the assay with human sera confirmed the repeatability, intermediate precision, and linearity of the assays; both homologous and heterologous specificity were verified as well; finally, limit of detection and quantification were established for all assays. Moreover, different sources of baby rabbit complement showed to have no impact on L-SBA output. The results obtained confirm the possibility of extending the L-SBA to multiple Shigella serotypes, thus enabling analysis of the functional response induced by natural exposure to Shigella in epidemiological studies and the ability of candidate vaccines to elicit cross-functional antibodies able to kill a broad panel of prevalent Shigella serotypes in a complement-mediated fashion. Full article

Five different silica nanoparticles functionalized with vitamin B12, a derivative of coumarin found in green plants and a minimum content of an organotin(IV) fragment (1-MSN-Sn, 2-MSN-Sn, 2-SBA-Sn, 2-FSPm-Sn and 2-FSPs-Sn), were identified as excellent anticancer agents against triple negative breast cancer, one of the most diagnosed and aggressive cancerous tumors, with very poor prognosis. Notably, compound 2-MSN-Sn shows selectivity for cancer cells and excellent luminescent properties detectable by imaging techniques once internalized. The same compound is also able to interact with and nearly eradicate biofilms of Staphylococcus aureus, the most common bacteria isolated from chronic wounds and burns, whose treatment is a clinical challenge. 2-MSN-Sn is efficiently internalized by bacteria in a biofilm state and destroys the latter through reactive oxygen species (ROS) generation. Its internalization by bacteria was also efficiently monitored by fluorescence imaging. Since silica nanoparticles are particularly suitable for oral or topical administration, and considering both its anticancer and antibacterial activity, 2-MSN-Sn represents a new dual-condition theranostic agent, based primarily on natural products or their derivatives and with only a minimum amount of a novel metallodrug. Full article

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. Full article

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. Full article

The temporary or permanent river blocking event caused by mass movement usually occurs on steep terrain. With the increase of mountain population and land use pressure and the construction of water conservancy and hydropower projects, river blocking events have gradually attracted people’s attention and understanding. The area in this study is affected by strong tectonic activity in the Jinsha River suture zone and the rapid uplift of the Tibetan Plateau. In the past 6000 years, there have been at least five obvious river blocking events in the reach. The number and density are very rare. Combining field investigation, indoor interpretation, laboratory tests, optically stimulated luminescence (OSL) dating, SBAS-InSAR and previous studies, multidisciplinary approaches are used to systematically summarize the analysis methods and further the understanding of one river blocking event and multiple river blocking events from different perspectives. Especially in multiple river blocking events, we can get the wrong results if interaction is not considered. Through this study, the general method of analyzing the river blocking event and the problems that should be paid attention to in sampling are given, and relatively reliable historical results of river blocking events are obtained. This method has applicability to the identification and analysis of river blocking events and age determination of dams with multiple river blockages. Full article

Serum bactericidal assay (SBA) is the method to investigate in vitro complement-mediated bactericidal activity of sera raised upon vaccination. The assay is based on incubating the target bacteria and exogenous complement with sera at different dilutions and the result of the assay is represented by the sera dilution being able to kill 50% of bacteria present in the inoculum. The traditional readout of the assay is based on measurement of colony-forming units (CFU) obtained after plating different reaction mixes on agar. This readout is at low throughput and time consuming, even when automated counting is used. We previously described a novel assay with a luminescence readout (L-SBA) based on measurement of ATP released by live bacteria, which allowed to substantially increase the throughput as well as to reduce the time necessary to perform the assay when compared to traditional methods. Here we present a further improvement of the assay by moving from a 96-well to a 384-well format, which allowed us to further increase the throughput and substantially reduce costs while maintaining the high performance of the previously described L-SBA method. The method has been successfully applied to a variety of different pathogens. Full article

Vi-polysaccharide conjugate vaccines are efficacious against typhoid fever in children living in endemic settings, their recent deployment is a promising step in the control of typhoid fever. However, there is currently no accepted correlate of protection. IgG and IgA antibodies generated in response to Vi conjugate or Vi plain polysaccharide vaccination are important but there are no definitive protective titre thresholds. We adapted a luminescence-based serum bactericidal activity (SBA) for use with S. Typhi and assessed whether bactericidal antibodies induced by either Vi tetanus toxoid conjugate (Vi-TT) or Vi plain polysaccharide (Vi-PS) were associated with protection in a controlled human infection model of typhoid fever. Both Vi-PS and Vi-TT induced significant increase in SBA titre after 28 days (Vi-PS; p < 0.0001, Vi-TT; p = 0.003), however higher SBA titre at the point of challenge did not correlate with protection from infection or reduced symptom severity. We cannot eliminate the role of SBA as part of a multifactorial immune response which protects against infection, however, our results do not support a strong role for SBA as a mechanism of Vi vaccine mediated protection in the CHIM setting. Full article

Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing. Full article

Development of a vaccine to limit the impact of antibiotic resistant Neisseria gonorrhoeae is now a global priority. Serum bactericidal antibody (SBA) is a possible indicator of protective immunity to N. gonorrhoeae, but conventional assays measure colony forming units (CFU), which is time-consuming. A luminescent assay that quantifies ATP as a surrogate measure of bacterial viability was tested on N. gonorrhoeae strains FA1090, MS11 and P9-17 and compared to CFU-based readouts. There was a linear relationship between CFU and ATP levels for all three strains (r > 0.9). Normal human serum (NHS) is a common source of complement for SBA assays, but needs to be screened for non-specific bactericidal activity. NHS from 10 individuals were used for serum sensitivity assays—sensitivity values were significantly reduced with the ATP method for FA1090 (5/10, p < 0.05) and MS11 (10/10, p < 0.05), whereas P9-17 data were comparable for all donors. Our results suggest that measuring ATP underestimates serum sensitivity of N. gonorrhoeae and that the CFU method is a better approach. However, mouse anti-P9-17 outer membrane vesicles (OMV) SBA titres to P9-17 were comparable with both methods (r = 0.97), suggesting this assay can be used to rapidly screen sera for bactericidal antibodies to gonococci. Full article